FACTOR 2024

Knowledge-driven Artificial Intelligence for Drug Discovery

Traditional discovery often takes 10–17 years and costs ~$2 billion to bring a drug to market, with a high attrition rate of 90%. On the other side, the world now faces a technological revolution in artificial intelligence (AI). AI machines can analyze huge databases of chemical, biological, medical, and research papers to find potential drugs for a given disease. Currently, close to 150 start-up companies are actively exploring AI technologies for drug discovery. Most of these contemporary data-driven AI technologies involve deep learning and other forms of machine learning systems, which tend to be “black boxes” that offer no insight into how they make their decisions and, therefore face challenges concerning transparency, trust, robustness, and explicability. Different from existing data-driven, black-box AI technologies for drug discovery, we are developing a knowledge-driven explainable AI-human-animal reinforcement learning drug discovery approach. I will discuss its applications in drug discovery for various diseases including Alzheimer’s disease, drug addictions, and cancer. I will also discuss its potential in drug discovery for osteosarcoma.

The Use of Weakly-Supervised Deep Learning for Necrosis Assessment in Patients with Osteosarcoma

Background: Percent necrosis (PN) after neoadjuvant chemotherapy is a prognostic factor for survival in osteosarcoma (OS). Currently, the assessment of PN is calculated by musculoskeletal (MSK) pathologists estimating tumor viability over an average of whole slide images (WSI). This process is labor intensive, requires subspecialized training, has high inter- observer variability, and is not standardized. Questions/Purposes: We aim to develop a machine learning model capable of 1) segmenting viable tumor, necrotic tumor, and non- tumor tissue with minimal training data and, 2) automatically calculating PN from OS WSI at comparable accuracy and precision to standard practice. Methods: We performed a proof-of- concept study and retrospectively obtained six WSI of patients with high-grade, intramedullary osteosarcomas at our institution. A weakly-supervised learning model was trained by utilizing coarse and incomplete annotations of viable tumor, necrotic tumor, and non-tumor tissue in WSIs by an MSK pathologist. Once trained, the model segmented areas of tissue and automatically determined PN from each WSI. To compare the fidelity of our machine learning model, an MSK pathologist estimated PN of each slide. We compared the machine model and pathologist PN estimates by Pearson’s correlation and mean absolute error (MAE). Results: Six WSI were segmented into areas of viable tumor, necrotic tumor, and non-tumor by the machine learning model and the MSK pathologist. The MAE was 14.8% over the six samples, but 6.4% when one outlier sample was removed, where the algorithm did not accurately label cartilaginous tissue. There was a strong, positive correlation between the machine system and MSK pathologist PN estimations by way of Pearson’s correlation (r=0.846). Conclusions: We were able to create and train a weakly-supervised machine learning model to automatically segment viable tumor, necrotic tumor, and non-tumor and calculate PN. This study also demonstrated a strong, positive correlation compared to the gold standard MSK pathologist PN estimation. We expect improvement can be obtained by annotating cartilaginous and other mesenchymal tissue for better representation of the histological heterogeneity in osteosarcoma.

Pixels to Predictions: Developing Imaging AI for Precision Oncology

Developing artificial intelligence (AI) schemes to assist the clinician towards enabling precision medicine approaches requires development of objective markers that are predictive of disease response to treatment or prognostic of longer-term patient survival. The solutions being developed in my group in this regard involve designing computational imaging features together with histology or molecular data for detailed tissue and disease characterization in vivo as well be associated with patient outcomes. The key innovation in this approach lies in “handcrafting” unique tools that can capture biologically relevant and clinically intuitive measurements from routinely acquired imaging (MRI, CT, PET) or digitized images of tissue specimens. Further, by conducting cross-scale associations between imaging, pathology, and -omics, we can not only “unlock” and integrate the information captured by these different, disparate data modalities but also develop an interpretable and intuitive understanding of what drives their performance. Specific problems in oncology being addressed via the new computerized imaging markers we have developed include: (a) predicting response to treatment to identify optimal therapeutic pathways, as well as (b) evaluating therapeutic response to guide follow-up procedures.

Envisioning Digital Twins for Cancer

Precision medicine is undergoing a transformation as the promise of virtual human models and personal medical digital twins is grabbing the attention of caregivers and researchers alike. This transformation builds on several key pillars including the ability to both collect, analyze, and share large amounts of data, innovative uses of AI, ready access to computing, and key insights from other industries such as transportation and aeronautics where digital twins have had success. The presentation will provide some background on digital twins, capabilities being developed, and how digital twins are being applied in biomedical applications including cancer. The essential role of the individual will also be explored in advancing research and developing new treatment choices as patients, caregivers, and researchers all work together to improve outcomes.

Engineered Bone Marrow as a Clinically Relevant Ex Vivo Model for Primary Bone Cancer Research and Drug Screening

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-year survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. To address this challenge, we adapted a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We formed eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 weeks for study. We tested ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, cultured OS cells within these constructs, and compared them to human OS samples. We showed that eBM stably recapitulates the composition of native bone marrow. OS cells exhibited differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we illustrated the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and showed that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

Validating Preclinical Models of Osteosarcoma Using Multiscale Transcriptomics‍

Preclinical models, such as cell lines and patient-derived xenografts (PDX), are an invaluable resource to better understand the biology of cancer and to test and develop new treatments. However, the drastic changes in environment involved in generating these models that enable them to survive outside of the human body can alter the characteristic gene expression program which drives the cancer, potentially voiding their resemblance to the disease which they model. Our group has developed both a multiscale atlas of cancer based on gene expression and a classifier, called OTTER, to match new patients and samples to the many cancer classes we found. We have previously reported four novel subtypes of osteosarcoma, each representing a distinct aspect of osteosarcoma biology, and which can prognosticate patients. To determine how well preclinical models of osteosarcoma match to primary disease in patients, we used OTTER to classify several commercial and patient- derived cell lines, as well as PDX’s, to our osteosarcoma classes. We report variable resemblance of these models to primary disease. We also report success in classifying murine models of other sarcoma types to human sarcoma classes. We are now focusing on expanding our tools to include a wider breadth of mesenchymal tumours, as well as releasing OTTER as a community resource. Our tools are publicly available, and we welcome continued engagement with clinicians and researchers to classify ongoing patients and preclinical models.

3D Collagen Culture Induces Osteosarcoma Chemoresistance through Drug Efflux Pump Overexpression

Treatment strategies for osteosarcoma (OS), the most common primary bone tumor in children and teenagers, have remained unchanged over the last 40 years. Current treatment involves chemotherapy and surgery, but this regimen causes significant side effects, and 5- year survival rates remain poor. One significant limitation in improving our treatment regimens is an incomplete understanding OS response to its extracellular matrix (ECM). This support structure provides a network of proteins and carbohydrates that cause biochemical signaling to direct OS growth and response to therapy. This work seeks to understand cell- matrix interactions by studying OS cells in a three-dimensional tissue-engineered matrix composed of collagen, the primary protein in the bone ECM. We hypothesized that cellular drug pumps that remove chemotherapy from cells are increased in 3D culture in a collagen matrix. We created a 3D tissue-engineered model of OS by embedding cells within a collagen hydrogel. We investigated how cells appeared, if they remained alive, and how they responded to chemotherapy. We then examined gene and protein expression of cellular pumps that remove chemotherapy. Encapsulated cells remained alive in 3D culture, grouped together and spread out in collagen matrices. When we treated 3D-cultured OS cells with chemotherapy, we observed less cell death compared to those cultured in a 2D environment. This suggests that the 3D-cultured cells may be more resistant to chemotherapy. Additionally, we found increased gene and protein expression of drug efflux pumps in 3D. These findings suggest OS cells grown in a 3D collagen environment become more resistant to chemotherapy by higher levels of drug efflux pumps. By using this three-dimensional culture system, we hope to uncover new ways to treat osteosarcoma and improve the dismal survival rates for patients.

Genomic Profiling and Modeling of Osteosarcoma Development in Murine Chondrocyte- Origin Models‍

Osteosarcoma (OS) is a primary bone tumor mainly affecting teenagers and young adults. Despite extensive research, the 5-year survival rate for metastatic OS remains low. Our mission is to develop more sensitive methods for detecting early metastatic cells to improve survival rates. Our research focuses on the origins and spread of OS, especially its migration to the lungs. We're investigating the source of spreading cells, their movement timing, and interventions to stop their progression. We're also looking for markers indicating early lung presence. OS typically arises during teenage years, a period of rapid bone growth. Growth plates at long bone ends are crucial for this growth. We suspect OS may start in these cells. We've genetically engineered mice, deleting genes p53 and Rb1 in growth plate cells, tagged with red fluorescence to track their development. These mice develop OS in human-like areas, supporting our hypothesis. Deleting these genes post-growth spurt reduces cancer incidence, linking bone growth and OS. Our mouse models mimic human OS in tumor appearance, genetics, and drug response, suggesting growth plate cells as the OS source. We've isolated pre-tumor fluorescent cells for genetic analysis to identify OS-initiating mutations. The fluorescence allows early lung metastasis detection. Our research delves into how these early cells adapt to the lung environment and the immune response they trigger. We aim to understand the immune changes during cancer progression. This work with engineered mice seeks to decode OS's complexities, leading to innovative treatments and improved OS patient outcomes.

Engineering Complex Models of Osteosarcoma: Unravelling Tumour Microenvironment Interactions for Drug Discovery and Therapeutic Innovation

Osteosarcoma, a highly aggressive bone cancer affecting children, adolescents, and young adults, presents a significant treatment challenge. The standard-of-care treatment plan has remained largely unchanged for nearly 50 years. Currently, secondary lung metastasis is the most critical clinical factor, with 70% of those affected succumbing to the disease within 3 years. Accelerating cures for patients with poor outcomes remains challenging, partly due to osteosarcoma's rarity. Therefore, recruiting enough eligible participants for clinical trials is a difficult endeavour. Traditionally, 2D cell cultures have served as the gold standard in vitro model in cancer research. While inexpensive and relatively easy to generate and maintain, they fail to accurately reflect the solid tumour characteristics and the complex crosstalk between tumour cells and their microenvironment. This persistent limitation has hindered the translation of preclinical drug candidates into successful clinical treatments. Recently, there has been increasing interest in developing more complex models for cancer research as they more closely mimic the phenotypic behaviour of original tumours compared to conventional 2D cell cultures. This is particularly crucial for sarcomas like osteosarcoma, where growth rates, cell morphology, cell-cell junctions, and kinase activation of spheroids have been shown to closely resemble those of primary tumours. This talk will discuss the different models of osteosarcoma and its interactions with other key players in the tumour microenvironment that we have developed in our lab to better understand disease progression and advance novel therapeutics.

Leveraging patient-derived osteosarcoma organoids for precision medicine

Patient-derived tumor organoids (PDTOs) are lab-grown replicas of tumors that closely resemble the original cancer in both structure and behavior. These models can be rapidly created from small samples obtained during biopsies or surgeries, allowing us to test their responses to different drugs in the lab. This makes them highly valuable for developing personalized treatment strategies, particularly in light of a growing body of evidence showing how PDTOs can in many cases accurately mimic clinical responses. Our team has developed a unique platform to grow these organoids efficiently from osteosarcoma surgical samples. We can create these three-dimensional avatars without needing complicated cell sorting or lengthy lab procedures. This means we can start testing drugs and get results within a week of the surgery, a turnaround that is rapid enough to potentially be used in the future to help guide treatment decisions. In a pilot study, we tested PDTOs from over one hundred sarcoma patients, including close to 30 osteosarcomas. We found that testing drugs on these organoids provided insights that closely matched the patients' actual clinical outcomes. We established organoids from biopsies of newly diagnosed osteosarcoma patients and tested the standard-of-care chemotherapy they were about to start. Organoids with low viability after chemotherapy matched patients who experienced higher tumor cell death (necrosis) after treatment and long-term no evidence of disease. In contrast, organoids more resistant to chemotherapy correlated with lower tumor response and quick disease recurrence. Additionally, we investigated responses of PDTOs from advanced, recurrent, and metastatic sarcomas. We found that the viability of these organoids correlated with the time to next treatment (TTNT) in patients. More effective multi-drug combinations on PDTOs resulted in longer TTNT, suggesting that these treatments were more successful in controlling the disease. Our data suggests that osteosarcoma organoids could help predicting how well some treatments may work. This approach complemented genetic testing and could be potentially leveraged to guide therapy. Encouraged by these promising results, we have initiated a clinical trial to further explore the use of PDTOs to predict osteosarcoma therapy responses (Clinical Trial: PREMOST, NCT06064682).

Leveraging Liquid Biopsy for Early Detection of Treatment Resistance and Metastasis

The ability to identify patients likely to experience early treatment failure and disease progression soon after diagnosis is a persistent challenge in osteosarcoma (OS). Liquid biopsy tests to detect cell-free DNA (cfDNA) can be used to help monitor patients for relapse. However, beyond detection of cancer, these approaches have not identified genetic changes that drive treatment resistance. Using dogs with naturally occurring OS, we sought to improve upon the accuracy of liquid biopsy by evaluating multiple genetic parameters in cfDNA to identify early signs of treatment resistance. We analyzed a combination of cryopreserved plasma samples from 17 dogs obtained throughout treatment, as well as samples collected from a prospective pilot neoadjuvant chemoimmunotherapy intervention study. Enrollment in this clinical trial completed two months after opening. Half of the dogs enrolled experienced early disease progression, enabling assessment of genetic changes associated with early relapse. Plasma samples were analyzed at intervals of 1-4 weeks from the time of diagnosis until disease progression was noted. cfDNA is being analyzed via whole methylome sequencing and ultra-low pass whole genome sequencing. Copy number changes in the plasma of dogs were identified one-month prior to clinical development of metastasis, showing that copy number alterations can be recognized prior to clinical metastasis. Investigation of additional timepoints prior to the development of metastatic disease are ongoing, in addition to integration of methylation changes to identify prognostic biomarkers within the first two months after diagnosis are underway.

Determinants of Metastatic Progression in Canine Osteosarcoma - A Translational Patient Animal Model for Humans‍

Osteosarcoma is a highly metastatic primary bone tumor that occurs spontaneously in both pet dogs and humans. Patterns of metastasis to organs beyond the most common site (lung) are poorly characterized and it is unknown whether specific associations between patterns of metastatic progression and patient features exist. This retrospective study characterized the necropsy findings of 83 dogs receiving standardized therapy and clinical monitoring in a prospective clinical trial setting to document patterns of metastasis and correlate outcomes with these patterns and other patient and tumor-specific factors. A total of 20 different sites of metastasis were documented, with lung as the most common site, followed by bone, kidney, liver, and heart. Two distinct clusters of dogs were identified based on patterns of metastasis. There was no significant association between site of enrollment, trial arm, sex, serum alkaline phosphatase (ALP) activity, or tumor location and clinical outcomes. A second cancer type was identified at necropsy in 10 dogs (10/83; 12%). These data showcase the extensive nature of osteosarcoma metastasis beyond the lung and provide a benchmark for clinical monitoring of the disease. Further, this study provides insight into transcriptional features of primary tumors that may relate to a propensity for osteosarcoma metastasis to specific organs and tissues.

Enhancer Rewiring Drives Osteosarcoma Metastasis‍

Think of gene enhancers as switches that turn on or dial up gene expression. They are not genes themselves, but they are the key drivers of cell identity. In any given cell type, there could be tens of thousands or hundreds of thousands of gene enhancers that form a sort of circuitry in the cell. My research has studied the enhancers’ role in osteosarcoma metastasis. We found there is a broad rewiring or reprogramming of the cell that occurs as osteosarcoma gains the ability to metastasize, and that turns out to be a key driver of that ability. When we took a 10,000-foot view at the differences in the enhancer landscape in a non-metastatic osteosarcoma cell and a metastatic osteosarcoma cell, we saw that there were many changes in the genome of a metastatic osteosarcoma cell that ultimately change the genes that cell expresses. Looking more systematically, we found that there were specific, non-random areas with gained enhancer activity and lost enhancer activity. Those changes seemed to center on particular genes. We looked at genes in metastatic cells where we saw a lot of increased enhancer activity and we asked, “If we alter the function of that gene, or we knock the gene down, can we limit the osteosarcoma cell’s ability to metastasize?” The answer we found was yes. Not only are these changes happening in a selective way, but they are functionally critical to that cell’s ability to metastasize.

Combination Therapies Targeting Heterogeneity and Lung Environment in Metastatic Osteosarcoma

Our understanding of the processes that give rise to metastatic lesions has evolved significantly in recent years. We used to think that metastatic lesions arose from the clonal proliferation of a few privileged cells that had acquired the multiple traits needed for survival and growth within the lung. However, we now understand that metastatic lesions are proper organs that require the coordinated activities of different subtypes of tumor cells and the surrounding host cells, which often adopt highly abnormal behaviors. We have begun to understand some of these underlying processes, which has led us to consider new ways of targeting metastatic disease. As one example, we have shown that the tumor cells that survive the initial stresses encountered within the lung have unique characteristics that allow them to engage lung cells in back-and-forth signaling. This interaction causes the lung cells to behave as if they have been wounded, triggering a wound-healing reaction that leads to scar tissue formation in the environment surrounding the tumor cells. This scar-like matrix appears to be essential for the development of the lesions. When we block the wound- healing process, we prevent metastatic lesion formation in several different models of metastatic osteosarcoma. In another example, we have found that the tumor-lung interactions occurring within the earliest developing lesions cause the tumor cells to express very high levels of a specific pro-survival protein called MCL1. We can prevent metastatic disease from forming by blocking MCL1 activity using clinically relevant drugs. When we combine MCL1 inhibitors with chemotherapy, we cure a large percentage of the mice that started with established metastatic lesions. These two examples illustrate how a mechanistic understanding of different tumor cell roles and the interactions between distinct tumor cell subtypes and the surrounding environment can inform therapeutic strategies targeting metastatic disease. Further validation of these concepts should guide the incorporation of such strategies into clinical trials.

CRISPRi Screening to Identify Vulnerabilities in Metastatic Osteosarcoma

Most osteosarcoma patients have micrometastasis at diagnosis. MAP therapy is effective for some patients, but others relapse due to the persistence of metastatic cells resistant to therapy. Despite recent advances, there has been little progress in the identification of clinically actionable vulnerabilities in osteosarcoma and in understanding what factors promote tumor cell survival in metastatic sites. Defining what allows tumor cells to survive at the metastatic niche (commonly the lungs) is therefore a critical need. My research hypothesizes that the metastatic ability of osteosarcoma is likely due to the presence of a subset of cells within the tumor that has an intrinsic capacity to survive in the metastatic site. The major goal of my work is to understand the connection between intratumor heterogeneity and metastatic capacity in osteosarcoma, as well as the role of specific oncogenic alterations in these processes. I hypothesize that focused loss-of-function CRISPRi screening coupled with DNA sequencing in vivo could identify genes driving metastasis when paired with functional studies. To test this hypothesis, I have developed an in vivo model using a lentiviral custom library of sgRNAs targeting potential osteosarcoma metastasis-related genes derived from RNA-seq and ATAC-seq analyses. I have performed both in vitro and in vivo screens using three osteosarcoma cell lines: SJSA (commercial), OS152, and OS742 (PDX-derived cell lines). Genomic DNA was extracted from all samples and the sgRNAs were quantified by deep sequencing and analyzed to identify potential therapeutic vulnerabilities for metastatic OS patients. Long term, my objective is to contribute to the identification of new therapeutic targets that can lead to clinical trials and ultimately improvement in patient outcomes for metastatic osteosarcoma.

Mechanisms Underpinning Osteosarcoma Genome Complexity and Evolution

My laboratory focuses on the development of computational methods to study the DNA alterations underpinning cancer development and drug resistance. In this talk, I will present our recent work on the analysis of genome sequencing data from hundreds of paediatric and adult osteosarcomas. Specifically, I will describe a novel, recurrent type of mutation that underpins the onset and progression of >50% high-grade osteosarcomas. In addition, I will present the data showing that the degree of chromosomal losses in osteosarcoma cells is a strong prognostic indicator for high-grade osteosarcoma. Overall, I will describe the patterns of mutations in osteosarcoma and the molecular processes that lead to its development, with implications for osteosarcoma diagnosis, treatment and patient stratification.

Overcoming Osteosarcoma Chemoresistance by Understanding and Targeting Cellular Quiescence

Chemotherapy, the best available treatment used for patients with osteosarcoma, the most common bone cancer, is designed to kill rapidly dividing cancer cells. The response to this toxic treatment is however poor in nearly half of such patients, contributing significantly to why survival has not improved since the introduction of chemotherapy 40 years ago. The reasons for this failure have not been fully explained. We have found that osteosarcoma cells grown in the laboratory escape the action of chemotherapy by halting their divisions and ‘going to sleep’, that is, becoming ‘quiescent cells’. Quiescent cells are identified using proliferation assays and by the expression of specific genes, which we show by using osteosarcoma cell lines and patient-derived samples. Quiescence is an unstable state, and osteosarcoma cells can ‘wake up’ from their sleep when the level of the chemotherapy drug reduces. Investigating sleeping osteosarcoma cells could allow us to develop ways of preventing them from going to sleep, killing them while they sleep, or pulling them out of their sleeping phase so that they would re-gain sensitivity to chemotherapy. The identification of sleeping osteosarcoma cells can be exploited in the future to design biomarker(s) that identify these cells in patients and target them to improve chemotherapy response.

Collateral Sensitivity During the Evolution of Resistance in Osteosarcoma Cells

Improvements in osteosarcoma treatment have been stagnant. When disease recurs or is resistant to first-line chemotherapy, patient outcomes become significantly worse. New treatment modalities and strategies are needed. We are researching the potential of collateral sensitivity in osteosarcoma cell lines with the hopes to eventually translate our findings to better treat patients with osteosarcoma. Collateral sensitivity is the phenomenon in which the evolution of resistance to treatment creates new avenues for treatment. Simply put, when a cell becomes more resistant to one chemotherapy, it may become more sensitive to another chemotherapy. This phenomenon has been observed in bacteria and our laboratory has shown that states of collateral sensitivity exist in Ewing sarcoma cell lines. We are studying osteosarcoma cell lines to look for collateral sensitivity and then correlate the sensitivity with genetic signatures.

Identification of Circulating Immune Cell Signature as a Biomarker of Disease Response and Resistance in Patients with Relapsed Osteosarcoma Treated with Nivolumab and Regorafenib

Osteosarcoma is the most common bone malignancy in children and young adults; a devastating cancer for which outcomes have not improved for decades. Recently, two clinical trials demonstrated that a chemotherapy drug called regorafenib had some benefit in adult patients with osteosarcoma whose tumor was not responding to treatment (refractory) or had returned (recurrent). One of the ways that regorafenib works is by shutting down signals in the cancer cell that cause it to grow. Immunotherapy is a treatment that allows the body’s own immune system to fight diseases such as cancer. Immunotherapy drugs that specifically target the immune system (e.g., nivolumab) have not been very successful in osteosarcoma. In certain types of other cancers, immunotherapy has a better effect if it is given with a drug like regorafenib. We conducted a multi-site trial (SARC038) through the Sarcoma Alliance for Research Through Collaboration testing a novel combination of nivolumab plus regorafenib to see if it will work better than regorafenib alone in patients with refractory/recurrent osteosarcoma. SARC038 trial has several correlative biology studies to better understand reasons why a patient does or does not respond to the therapy. One of the biology objectives is a detailed analysis of immune cell components and immune-related gene expression profiles, which is akin to understanding the immune cells programming and instruction set. We will present our preliminary findings from the analysis of samples in the 23 patients enrolled in the first stage of the trial.

The role of aberrant MYC expression in osteosarcoma

The MYC gene is frequently disrupted in cancer where it is responsible for multiple malignant features including unchecked growth, metastasis and immune evasion. In osteosarcoma, MYC amplification (multiple copies of the gene) has been associated with metastasis and poor survival, suggesting that MYC inhibition would be beneficial. However, targeting MYC has been challenging, leading to research into therapeutic strategies that either inhibit its expression or hasten its degradation. We have reported on ways to curtail MYC RNA production through disrupting a set of molecules, transcription-associated CDKs, that are critical to gene expression, those that can perturb MYC protein expression, both of which need to be developed for clinical testing. Further studies are also needed to determine the exact function of aberrant MYC in osteosarcoma cells, whether MYC protein overexpression due to causes other than gene amplification is also associated with metastatic disease and treatment resistance or whether the prognostic effects are related to the amount of MYC (dosage) in the cells rather than the mechanism of overexpression. Answering these questions will be critical to the establishment of oncogenic MYC not only as a biomarker of metastasis, treatment response and resistance, but also as a therapeutic target in osteosarcoma.

Optimizing DNA Damage Repair Pathway Inhibition for Osteosarcoma Therapy

Osteosarcoma (OS) is the most common malignant bone tumor in children. Children with OS are treated with intensive myelosuppressive chemotherapy and aggressive surgical resection. Despite this, long-term survival is sub-optimal, and those who do survive struggle with multiple chronic sequelae of treatment, including hearing loss, cardiomyopathy, and orthopedic problems. Thus, it is critical that new, more effective treatment strategies are developed for OS. OS is characterized by countless somatic mutations and heterogeneous genomic copy number events and chromosomal rearrangements, making it an impressively heterogenous disease. We have observed, in genome-scale CRISPR screening in OS cell lines, that among the most selective survival genes (dependencies) in this disease there is a strong enrichment for DNA damage repair (DDR) genes. As DDR inhibitors are a group of therapeutics that have had marked successes in some adult tumor types, this has heightened our interest in these strategies to treat OS. PARP inhibitors, one of the most successful DDR classes of drugs, have shown mixed responses in OS patients and pre-clinical models, though the genomics of OS suggest specific features that lend them to PARP inhibitor sensitivity. We thus utilized an unbiased genomic approach to nominate synergistic druggable targets with PARP inhibitors in OS, CRISPR-Cas9 synergy screening. A top scoring screen target, ATM, is a druggable kinase with a role in the DNA damage response. We have investigated ATM plus PARP inhibition in vitro in OS cell line models and have found a high degree of synergistic cell death between the two classes of agents, as well as higher amounts of apoptosis and more DNA damage in the combination treated cells. We believe that this strategy may be highly efficacious for treating some or all OS and are moving forward to validating ATM and PARP inhibitors together in vivo in unique PDX models, with the ultimate goal of translating these findings to a clinical trial for human patients with OS.

Advancing Precision Oncology in Osteosarcoma: Novel Biomarkers for Enhanced Risk Stratification and Tailored Treatment Strategies

The goal of this study is to improve risk stratification in osteosarcoma in children, adolescents and young adults. Risk stratification means sorting patients into different risk groups based on the severity of their cancer, allowing doctors to ultimately tailor treatment to individual patients rather than using a one-size-fits all treatment approach. Currently, all patients diagnosed with osteosarcoma receive the same treatment, regardless of whether they have a high or low probability of being cured. This treatment, which has been used for over four decades, is very toxic and does not cure 30-40% of patients. Moreover, if osteosarcoma comes back or spreads to other parts of the body, it becomes very difficult to cure and is often fatal. Doctors currently don’t have good ways of predicting which patients will be cured from the standard treatment approach and which patients are likely to die from their disease. We believe that there are different groups of osteosarcoma with unique genetic features that affect how the cancer behaves in patients. We will study osteosarcoma tumors collected from patients to identify a genetic “signature” that can tell us which patients are likely to be cured from standard treatments and which patients are at high risk for dying from their osteosarcoma and may benefit from different treatments. We will then use this signature to develop an easy-to-use test that can be that can be used in patients to assess risk group in real time when patients are first diagnosed with osteosarcoma.This method could eventually help doctors choose better treatment for different groups of patients with OS, leading to new clinical trial trials that incorporate more personalized and effective care for osteosarcoma.

Pharmacokinetics, Pharmacodynamics and Tolerability of Vismodegib in Dogs with Spontaneous Osteosarcoma

Spontaneous canine osteosarcoma (OS) is regarded as the most faithful model of the human disease. The hedgehog (Hh) signalling pathway, important in development and altered in some human cancers, appears to contribute to human and canine OS progression, and metastasis inhibition has been observed with Hh inhibition in laboratory OS studies. Vismodegib is an FDA-approved Hh inhibitor. Previous studies have established a tolerable dose range of vismodegib in laboratory dogs; however, vismodegib has not been evaluated in dogs with spontaneous cancer. The goal of this study was to determine the tolerability, drug levels and biologic effects of vismodegib, when administered in conjunction with standard of care therapy in dogs with spontaneous OS. Dogs with OS of the limb underwent amputation, and 1 week later began treatment with vismodegib (150 mg daily). Carboplatin was started 2 weeks later and continued every 3 weeks for up to 6 treatments. Plasma drug levels were assessed using published methods. Skin biopsies were obtained at surgery and 2 and 8 weeks after starting vismodegib assessed for changes in GLI1, a marker of Hedgehog pathway inhibition. Adverse effects (AE) were graded using published Veterinary Cooperative Oncology Group (VCOG) criteria. 6 dogs have been enrolled to date. 5 were treated with continuous vismodegib and 1 received treatment on a 14 days on / 7 days off schedule. Median body weight was 38 kg (range 16.5 to 65.2 kg), equating to a median administered dose of 4.0 mg/kg (range 2.3 to 9.1 mg/kg). Drug levels were predicted to be associated with Hh inhibition in all patients. Significant (>75%) reduction in Gli1 was observed in skin biopsies from 3 of 5 evaluable patients. AE attributable to vismodegib were largely grade 1 and 2 and reversible and included liver enzyme elevation, reduced appetite, and cutaneous changes consisting of hair loss, redness and thickening of the skin of the nose. The hair loss noted with continuous dosing prompted the intermittent dosing in Dog 6. This dog experienced no significant skin changes. In conclusion, vismodegib appears to be tolerable and associated with favorable drug levels and biologic effects in dogs with spontaneous OS undergoing standard of care therapy. Future studies will evaluate the effect of vismodegib co- administration on outcome in dogs with appendicular OS.

Tumor-intrinsic cGAS-STING Activation Promotes Anti-tumor Inflammatory Response in Osteosarcoma

This work will identify ways to activate the immune system to fight osteosarcoma, which is critically important to improve the treatment options available for children affected by this disease. Doctors and researchers have known for some time that our immune system can recognize cancer and remove it. In fact, many new cancer treatments have used this “immunotherapy” approach, but we haven’t yet figured out how osteosarcoma hides from the immune system or how to reverse this. In this project, I have identified that the cGAS- STING pathway allows osteosarcoma to avoid the immune system, and I am now testing a new approach of activating STING to unleash the immune system to treat osteosarcoma. We believe this research will result in improved understanding of how osteosarcoma avoids immune surveillance, and in a novel treatment paradigm for this highly aggressive pediatric cancer. I am very excited to continue this work which I think in the next few years could result in a new treatment for patients with osteosarcoma. Recent attempts to introduce better treatments for osteosarcoma have not been successful, and this data can serve as the basis for the development of a broad range of new immunotherapy treatments for this disease.

Immunosuppressive Tumor Microenvironment in Osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Overall survival of osteosarcoma patients has not significantly improved in last 40 years, and a large portion of patients die from the effects of distant metastatic tumors, particularly metastases in the lung. Five-year survival rate of patients with localized disease is ~70%, while survival decreases to 20-30% for the patients with metastasis. Recent clinical trials of immunotherapy for osteosarcoma patients with metastatic or unresectable primary tumors did not show beneficial results. To address the need to improve the efficacy of immunotherapy for osteosarcoma patients, we attempt to use single cell and spatial biology techniques to dissect the osteosarcoma tumor microenvironment of primary and metastatic tumors, with a focus on its immune components that are likely undermining immunotherapy via immunosuppression. Previous transcriptomic analyses of osteosarcoma tumors by our group have revealed an abundance of cells from the myeloid lineage in the tumor microenvironment, including macrophages, monocytes, neutrophils, and dendritic cells. Through single-cell tran¬scriptomic analysis of diagnostic biopsies of primary osteosarcoma tumors, we found a subpopulation of immunosuppressive neutrophils that express markers of previously described myeloid-derived suppressor cells, which have been shown to suppress the adaptive immune responses in cancers. We are now trying to understand how these immunosuppressive neutrophils contribute to the immunosuppressive tumor microenvironment and promote tumor development in the bone.

Osteosarcoma Stratification and Subtyping with Multiomics Approaches

Up to now, Osteosarcoma stratification only relies on clinical parameters and histological response. Over the last years, our team build up a new arsenal of prognostic factors based on the molecular profiling of Osteosarcoma tumors and the analysis of circulating DNA and protein molecules. Still at the pre-clinical stage, this new generation of prognostic factors or classifications might quickly improve the cure of patients by an early orientation to suited chemotherapies and the identification of new therapeutic agents matching their molecular profiling. In order to accelerate the translation into new clinical practices, we investigated the molecular profiles of patients to better understand the tumor biology and to identify vulnerabilities in hard to treat patients.

Doing a Lot from a Little: Multi-omic Profiling of Metastatic Osteosarcoma‍

Tumors are tissues, and like any other tissue in the body, tumor tissues are made up of different cells and molecules. To understand how these cells and molecules work together to help the tumor grow and resist therapy, we need to deconstruct the tumor tissues cell-by-cell and molecule-by-molecule. Traditionally, this has required a lot of tissue, which is often hard to get, and resulted in just a few data points. Recent advances in technology has drastically changed this. Our lab has established a workflow that allows us to understand what individual cells and small groups of cells are doing, how they communicate, how they are arranged, and determine the function of these cellular neighborhoods at both the protein and RNA level. Rather than requiring significant amounts of tumor tissue, we can perform most of this analysis on a mere handful of slides and small pieces of frozen tissue, allowing us to examine tumors in great detail without depleting the tissues so that they can be used by other investigators for other purposes. Here, I will share some of what we learned and how we plan to use this strategy going forward.

Mapping the Single-cell Differentiation Landscape of Osteosarcoma

High-grade osteosarcoma (OS) is an aggressive and heterogeneous disease that can present with diverse tissues features resembling several mesenchymal cell types, including bone and cartilage. Nevertheless, current OS treatment kills all proliferating cells similarly without addressing the root disease cause: dysregulated differentiation. This uniformity arises from limitations in understanding both how mesenchymal cells types are driven to their terminal cell fates and how OS dysregulates this process. To pinpoint genetic signatures of OS lineage heterogeneity, we transcriptionally profiled 31,527 cells from a tissue-engineered model, enabling us to characterize, for the first time, the continuum of transcriptional states preceding terminal adipogenic and osteoblastic differentiation. Incorporating pre-existing chondrocyte data, we applied a mathematical clustering technique to generate the first time- resolved human atlas of mesenchymal differentiation states. This transcriptional 'roadmap' served as a reference to delineate the single-cell composition of morphologically complex OS tumors and identify gene modules linked to OS cell fate and patient survival. Our study takes the critical first step in accurately quantifying OS differentiation and lineage, enabling a novel, differentiation-based drug screening strategy.

Osteosarcoma Explorer, a Data Commons with Clinical, Genomic, Protein, and Tissue Imaging Data for Osteosarcoma Research

Osteosarcoma research advancement requires enhanced data integration across different modalities and sources. Current osteosarcoma research, encompassing clinical, genomic, protein, and tissue imaging data, is hindered by the siloed landscape of data generation and storage. Here, clinical, molecular profiling, and tissue imaging data for 573 patients with pediatric osteosarcoma were collected from four public and institutional sources. A common data model incorporating standardized terminology was created to facilitate the transformation, integration, and load of source data into a relational database. On the basis of this database, a data commons accompanied by a user-friendly web portal was developed, enabling various data exploration and analytics functions. The Osteosarcoma Explorer (OSE) was released to the public in 2021. Leveraging a comprehensive and harmonized data set on the backend, the OSE offers a wide range of functions, including Cohort Discovery, Patient Dashboard, Image Visualization, and Online Analysis. Since its initial release, the OSE has experienced an increasing utilization by the osteosarcoma research community and provided solid, continuous user support. To our knowledge, the OSE is the largest (N = 573) and most comprehensive research data commons for pediatric osteosarcoma, a rare disease. This project demonstrates an effective framework for data integration and data commons development that can be readily applied to other projects sharing similar goals. The OSE offers an online exploration and analysis platform for integrated clinical, molecular profiling, and tissue imaging data of osteosarcoma. Its underlying data model, database, and web framework support continuous expansion onto new data modalities and sources.

Flash Radiotherapy: Current Work and Future Directions

Radiation therapy is an important part of treatment for many pediatric cancers including sarcoma, but there remains a need to improve both tumor control and side effect profiles from radiotherapy. Flash therapy is an investigational approach to radiation that delivers treatment at ultra-high dose rates compared to standard radiation. In initial studies in cell lines and animals, this resulted in similar tumor control with fewer side effects than current approaches to radiotherapy. It is currently under study in human patients with bone metastases and the first clinical trial showed the feasibility and safety of proton-based Flash treatments, resulting in significant interest in evaluating its role in treating additional tumor types in the near future.

Multi-Site SBRT for Pediatric, Adolescent, and Young Adult Patients with Osteosarcoma: Local Failure Dosimetric Analysis

Stereotactic Body Radiation Therapy (SBRT) is a radiation technique that delivers high dose focused radiation in a limited number of treatments (fractions), ranging from 1-5. Osteosarcoma is traditionally thought of as a radiation resistant tumor which is why standard treatments include surgery and chemotherapy. However, given the increased use and familiarity of SBRT, SBRT may be a technique that can be utilized in osteosarcoma patients for focal ablation of recurrent disease or distant metastases. This current research evaluates patients treated with SBRT at the Cleveland Clinic over the past 8 years who had local failures, meaning those patients who had further progression of disease at the site treated with SBRT. We sought to categorize the failures as in-fieldmeaning most of the recurrent tumor was in the high dose radiation volume, marginal- meaning 20-80% of the recurrent tumor was in the high dose radiation volume, or out-of-field- meaning <20% of the recurrent tumor was in the high dose radiation volume. The goal was to report on common sites of failure and hopefully identify common reasons for failure. We identified 27 patients and a total of 78 lesions that were treated at Cleveland Clinic from 2015-2023. Most of the lesions (64%) were in the bone, but other sites included lung, liver, and soft tissue. Of all the bony sites, 54% of them were in the spine. The number of treatments varied from 1-5 and the dose delivered ranged from 16- 60 Gy. The median follow-up time from treatment to last local control image or to local treatment failure was 6.1 months. There was a total of 11 local failures (14%) and 4 of them were in-field, 3 were marginal, and 4 were out-of-field. The spine was the most common site of failure (n=7). Out of all lesions treated, SBRT to the spine was associated with the highest risk of failure at 26%, followed by soft tissue at 17%, and extremity at 8.3%. SBRT for osteosarcoma metastases is an effective local treatment with 86% local control in this series. Of the local failures, they were evenly distributed amongst in-field failures, marginal, and out-of-field local failures. In our series, SBRT to the spine had the highest risk of recurrence. This research is important as it may help guide radiation oncologists in the future in drawing treatment volumes, determining the appropriate radiation dose, and collaborating on ways to reduce treatment failures especially those in the spine.

Interventional Radiology Local Control Options in Osteosarcoma

Interventional oncology has emerged as the fourth pillar of cancer care, complementing medical, surgical, and radiation oncology. This field encompasses several minimally invasive procedures that target cancerous tumors with precision, often under image guidance. Despite their proven efficacy in tumor reduction, these techniques are not yet widely adopted. Key Procedures and Benefits: Thermal Ablation: Techniques such as radiofrequency ablation (RFA), microwave ablation (MWA), and cryoablation use extreme heat or cold to destroy cancer cells. These methods offer precise targeting, minimizing damage to healthy tissues. Transarterial Chemotherapy (TACE): This procedure delivers chemotherapy directly to the tumor via its blood supply, reducing systemic side effects. These modalities provide several advantages: Precision: Ensuring direct targeting of tumors. Minimally Invasive Nature: Resulting in quicker recovery times. Effective Local Control: Managing primary tumors and palliating symptoms. Repeatability: Allowing for repeated treatments if necessary. Combination Therapy: Enhancing the efficacy of systemic chemotherapy. Abscopal Effect: Certain ablative techniques, notably RFA and cryoablation, have demonstrated the potential to boost the body's immune response to cancer, a phenomenon known as the abscopal effect. This effect may aid in treating metastatic disease by prompting the immune system to target distant tumor sites. Supportive Procedures: Interventional oncology also includes crucial supportive procedures such as needle biopsies for diagnosis, port and line placements for chemotherapy administration, and drainage procedures to manage complications like fluid accumulation or air leaks. Application in Osteosarcoma: In the context of osteosarcoma, a primary bone cancer, interventional oncology can play a significant role in both treatment and palliation: Local Control: Thermal ablation and TACE can effectively reduce or eradicate tumors. Symptom Palliation: These techniques can alleviate pain and other symptoms associated with tumor burden. Immunologic Enhancement: Ablative procedures may enhance the immune response to metastatic spread. Collaborative Approach: Optimizing treatment outcomes necessitates a multidisciplinary approach involving interventional oncologists, medical oncologists, surgeons, and radiologists. Collaboration in clinical practice and research is vital for developing more effective management strategies for osteosarcoma and other cancers. Raising awareness among healthcare providers and patients about the benefits and availability of interventional oncology is essential for its broader adoption in cancer care. This approach promises precise, minimally invasive treatment options that can be combined with systemic therapies to enhance overall treatment efficacy and patient outcomes.

Separate Resection of Biopsy Tract and Primary Sarcoma: Implications for Local Recurrence and Overall Survival

Osteosarcomas are diagnosed with a biopsy, a small sample of the tissue in question that is removed for testing, and then treated with a combination of chemotherapy and surgery. Traditionally, at the time of surgery when the tumor is removed, the biopsy tract and the area around the biopsy is removed all at once as a large piece. Trying to leave the biopsy tract and the tumor connected can limit surgical options and can lead to compromises that may not have to be made. This study aims to test whether removing the biopsy tract separately vs with the tumor has an y impact on the tumor growing back in that area or on overall survival. We looked back at osteosarcoma cases from the last 10 years and compared those where the biopsy tract was sent separately to those more traditionally resected with the tumor. Our results indicate that removing the biopsy tract separately is safe and does not increase the chances of the tumor returning.

3D Virtual Planning and Patient-Specific Instrumentation to Improve Outcomes of Physeal-Sparing Intercalary Resections and Allograft Reconstructions

Surgical removal of the diseased area of bone in bone sarcoma is a standard component of treatment. Limb-sparing techniques have been developed to replace the removed bone with something else to keep the limb when possible. There are metal replacements, but when close to the ends of the bones, these sacrifice the native ligaments of the joint and eliminate the growth plate from where the body naturally makes the bone longer. This is a problem in young patients with a lot of growth potential left. Sometimes the tumor can be resected in such a way that the growth plate is preserved, and the diseased segment of bone is replaced with a donated segment of bone from a cadaver. In young patients, reconstructing the bone in this way can be challenging because there is limited space to work with to not damage the growth plate. Our study investigates our use of 3D virtual, and 3D printed models as well as 3D printed surgical bone cutting guides to help remove these tumors and reconstruct the bones with donor bone while maintaining the native growth of the bone. We have developed novel techniques to do this and customize reconstructions before surgery to ensure a perfect fit and a shorter operative time. Our results indicate that the time of surgery is reduced, the rate of repeat surgery is reduced, and that the fit of the native and donor bone as well as the rates of healing are all improved using these new 3D technologies.

Preclinical Evaluation of Folate Receptor Alpha-specific CAR T Cells Against Osteosarcoma

Osteosarcoma is the most common primary bone cancer in children and young adults. Unfortunately, metastatic or relapsed disease remains very difficult to treat with limited treatment options. Even more discouragingly, there has not been significant advances within the past several decades. New strategies need to be explored to find better, more effective treatments. Chimeric antigen receptor T cells (CAR T) are engineered immune cells that express a receptor that recognizes and kill tumor cells. Our lab has generated CAR T cells that are able to recognize and kill cells that express folate receptor-alpha, referred to as FOLR1. Previously published literature as well as public databases have shown that a majority (70- 80%) of patient-derived samples of osteosarcoma express FOLR1. Therefore, we hypothesize that CAR T cells engineered to recognize FOLR1 (FOLR1-CAR T cells) will be effective in eliminating osteosarcoma tumor cells. Our studies in the lab thus far show that FOLR1-CAR T cells are able to recognize and kill a standard osteosarcoma cell line in culture. We observed >90% tumor cell death when osteosarcoma cells were placed in culture with FOLR1-CAR T cells by 24 hours. We also tested FOLR1-CAR T cells in mice with human osteosarcoma implanted in them as a localized tumor as well as metastatic lung disease. In both models, we observed disease remission by imaging and prolonged survival in treated mice in comparison to untreated mice. We plan to complete similar studies using osteosarcoma cell lines derived from patient samples. Once these studies are complete, we plan to proceed with translating FOLR1-CAR T cells to be tested in human patients with relapsed or refractory osteosarcoma. We hope that this treatment option will not only provide disease response, but decrease short-term as well as long-term toxicities of treatment.

A Novel Dendritic Cell Vaccine Targeting CD70 for Osteosarcoma Lung Metastasis

There is an urgent need for new therapies to treat osteosarcoma metastases as survival rates remain < 20%. No effective therapies have been identified in >20 years. The activity of check- point inhibitors has been disappointing and 7 recent phase II trials from COG failed to identify new approaches. Dendritic cell vaccines (DCVs) are an emerging focus in immunotherapy with demonstrated clinical efficacy against melanoma and other solid tumors. There are different subsets of DCs that can be used to generate a DCV. Monocyte-derived DCVs (MoDCVs) are well tolerated in children and adolescents but have low therapeutic activity in OS. A DCV generated using cDC1 cells, (a unique subset of DCs that are essential in the anti- viral immune response in the lung) is more potent than the MoDCV. We showed that a DCV generated using cDC1 cells and OS tumor cell lysates induced the regression of primary OS tumors and lung metastases, and a systemic immune response that led to regression of untreated tumors. This provides proof of principle that cDCVs can be a novel immunotherapy against OS. However, cDCVs generated using tumor cell lysates are not optimal for clinical development as it requires obtaining a sample of the patient's tumor, lysing the tumor in the laboratory and culturing the tumor cell lysate with the patients cDCs to generate the cDCV. Such specialized techniques preclude the ability for widespread usage. Clinical translation requires the identification of a common OS antigen. The protein CD70 is overexpressed in OS but absent in normal cells making it a good therapeutic target. We generated a CD70-cDCV using CD70 recombinant protein which is readily available. We showed that the anti-tumor activity of the CD70-cDCV and its ability to generate an systemic anti-tumor response against both primary and OS lung metastases was equivalent to the cDCV generated using tumor cell lysates. The CD70-cDCV also prolonged survival similar to the lysate cDCV. These studies indicate that a CD70-targeted cDCV may provide a novel immunotherapeutic approach for relapsed OS patients with metastases in the bone and lung, thereby improving survival for this difficult patient population with few alternative therapies. Because this approach uses recombinant protein it does not require access to the patient's tumor, thereby simplifying therapy initiation.

Harnessing the Osteosarcoma Surfaceome for Immunotherapy Targets to Block Metastatic Capacity

With current treatment options, patients with Osteosarcoma have a 5-year survival rate of about 76%, but 3 out of 4 patients diagnosed with Osteosarcoma that has spread beyond the primary site will not live 5 years passed their diagnosis. Further, Osteosarcoma that returns or becomes resistant to conventional treatment remains a significant challenge. There is clearly an unmet clinical need to develop new options for these patients. Immunotherapy aims to notify your body of the malignant cells and target them for destruction by the patient’s own immune system, however, while success has been demonstrated in adult cancers and some pediatric blood cancers, its success in pediatric solid tumors is still lacking. One crucial aspect of developing successful immunotherapies is having a good target, and these are often targets that sit on the outside surface of the cancer cells. To address this, we have successfully characterized the outside surface of Osteosarcoma in a large cohort of patient samples. We have compared the Osteosarcoma surface to other cancer surfaces and identified a number of targets highly specific to Osteosarcoma, and these may represent actionable immunotherapies worthy of further investigation. In the future, we hope that our work can contribute to the development of a new strategy for Osteosarcoma patients.

Making Cellular Therapy Work for Osteosarcoma

Adoptive cell transfer (ACT) of genetically-engineered T cells has induced durable complete responses (CR) in patients with otherwise incurable and fatal cancers. The use of chimeric antigen receptors (CAR) for treating lymphoid malignancies is the most striking example, inducing high rates of durable CRs. However, the incredible outcomes seen using CAR T cells have not been replicated in solid tumors and this strategy generally fails to completely eradicate disease, producing only transient benefit. Several groups have tested chimeric antigen receptor (CAR) T cells in osteosarcoma (OS) however issues in the tumor immune microenvironment may be blocking their efficacy. We are currently exploring several potential resistance mechanism is osteosarcoma and developed a canine osteosarcoma CAR in order to examine these more closely.

Immune Profile and Functional Evaluation of Tumor-infiltrating Lymphocytes in OS Tumor Microenvironment

The immune system contains powerful cell types capable of fighting the tumor but the immune cell types within the tumor microenvironment in osteosarcoma can be quite different from patient to patient. In this study, we examined individual immune cell types within the tumor and expanded the tumor-infiltrating lymphocytes (TIL). TIL contain cells that are soldiers engaged with fighting the tumor by directly killing tumor cells. Understanding how TIL are able to function and what they are reacting to within the tumor microenvironment furthers our understanding of how to engage these powerful cells. In order to achieve this goal, we profiled the immune cells including TIL and other cell types called antigen presenting cells. Antigen presenting cells are able to instruct TIL into pro or anti-tumor functional states. In this cohort, we found that while the tumor microenvironment does not contain a lot of immune cells, some tumors do contain TIL and antigen presenting cells. However, the TIL express markers that suggest that they may be exhausted and suppressed within the tumor. Excitingly, when we expand the TIL, the cells are able to make proteins that are used to kill tumor cells. Next steps involve further characterizing these TIL to define if they can directly kill tumor cells.

CAR T Cell Immunotherapy for Osteosarcoma

The prognosis for patients with relapsed osteosarcoma is poor. Although there are several agents with activity in recurrent or refractory osteosarcoma, there are no medical therapies proven to improve overall survival in this population. The potential of using immune-based therapeutics is attractive because of the opportunity to invoke anti-cancer mechanisms to which chemotherapy/radiation-resistant tumor cells may be susceptible, and because of limited toxicity theoretically possible with tumor-specific immunologic targets. The recent success of CD19-specific chimeric antigen receptor (CAR) T-cell therapy in high-risk B-cell malignancies has generated interest in investigating this approach in solid tumors. Many challenges remain in optimizing CAR T cell therapies and solid tumors pose unique obstacles for the success of this strategy including selection of an optimal target, effective trafficking to tumor sites, and proliferation in the immunosuppressive tumor microenvironment. Seattle Children's Therapeutics has developed multiple CAR T cell trials for patients with solid tumors, including osteosarcoma, that attempt to address some of these barriers while providing unique experimental treatment options to patients who have failed standard curative therapies. The STRIvE trials include an arm employing treatment with tumor-directed and anti-CD19 (bispecific) CAR T cells in an attempt enhance expansion and trafficking to tumor sites, while the ENLIGTHen-01 trial utilizes intrapatient escalation of a CAR adapter molecule (CAM) to target the folate receptor in osteosarcoma.

Outcomes for osteosarcoma have remained essentially unchanged for several decades, despite numerous attempts to improve survival rates through intensification of therapy or the addition of more chemotherapy drugs based on treatment response. While a number of new types of therapies have been studied with little success, several agents from the drug class of multi-targeted tyrosine kinase inhibitors (MTKIs) have shown evidence of activity in patients with advanced or relapsed osteosarcoma. MTKIs are now commonly used as a second line treatment for patients with relapsed disease, but their role in the upfront setting is not yet known. MTKIs have unique potential side effects that must be considered when deciding how these agents may be able to be safely given along with chemotherapy. This presentation will overview the development and trial design of AOST2032, a currently active Children's Oncology Group clinical trial that will first assess the safety and feasibility of combining an MTKI with standard chemotherapy agents in patients with newly diagnosed high-risk osteosarcoma. This study is the first national clinical trial for newly diagnosed osteosarcoma to open in 20 years, and presents an exciting opportunity to improve outcomes for this population.

The Osteosarcoma Count Me In Project of the Participant Engagement Cancer Genomic Sequencing Network: Directly Engaging Patients in Genomic Research

Osteosarcoma (OS) is a sarcoma with complex genomes for which there has been limited progress in identifying new treatments and improving outcomes. Slow progress in OS is partly due to insufficient characterization of the genomic landscape. Generating genomic datasets in OS is challenging due to the rarity of these sarcomas and recruitment barriers like care fragmentation between institutions and specialties. Count Me In, a research initiative with prior success in angiosarcoma, worked with patients and advocates (including MIB Agents) to create a website (OSProject.org) where patients register and consent to participation. The OSProject (OS1) launched in February 2020 and then re-launched (becoming OS2) after receiving National Cancer Institute Funding and becoming part of the PE-CGS Network in September, 2022. 137 patients aged 5-63, receiving care from 70+ institutions, had consented to the OS1 Project (prior to the relaunch). 118 patients aged 7-74, receiving care from over 65 institutions, have consented to the OS2 Project (a part of the PE- CGS Network). 61 participants from OS1 reconsented to OS2, for a total of 255 Count Me In OS participants. Blood and saliva are collected from consented participants, tumor samples are obtained from pathology departments and medical records are requested from hospitals. WES and WGS of tumor and normal, and RNASeq of tumor is performed. ctDNA is obtained and sequenced. Results are shared with patient, advocacy, physician and research communities in several ways. Individual participants receive a shared learning report describing the somatic variants identified in their tumor from paired tumor-normal WES and are offered genetic counseling and clinical germline testing. Registered participants receive updates via email and Project websites.

Phase 1 Study of HS-20093, a B7-H3 Targeting Antibody-Drug Conjugate (ADC) in Relapsed or Refractory Osteosarcoma and Other Sarcomas (ARTEMIS-001)

A Beacon of Hope for Advanced Sarcoma Patients Patients with relapsed and refractory (R/R) sarcomas face a significant medical challenge, and the ARTEMIS-001 study aimed to address this unmet need. Focusing on the overexpressed B7-H3 protein in sarcomas, a novel treatment, HS-20093, emerged as a beacon of hope. In this groundbreaking phase 1 trial, 9 patients with R/R sarcomas, including osteosarcoma and various soft tissue sarcomas, received intravenous HS-20093. Doses ranged from 4.0 mg/kg to 16.0 mg/kg, revealing promising results. The study showcased HS-20093's potential. Safety was a priority, and all patients experienced treatment-related adverse events, with 66.7% facing grade 3/4 events, primarily hematotoxicity. Despite these challenges, the safety profile aligned with expectations. Of the 9 evaluable patients, two showed confirmed partial responses at 4.0 and 8.0 mg/kg HS-20093, yielding an overall response rate of 22.2%. This positive outcome occurred at the initial efficacy assessment. Impressively, the disease control rate reached 88.9%, demonstrating HS-20093's ability to manage and stabilize the disease. The patient with the longest treatment duration, an outstanding 330 days, remained on HS-20093 until the last follow-up in July 2023, emphasizing its potential for sustained benefits. The 4-month progression-free survival rate was 89%. In conclusion, HS-20093 exhibited highly promising antitumor activity, particularly in R/R osteosarcoma and other sarcomas that had progressed on standard therapy. This success has propelled the initiation of a phase 2 study, currently underway (NCT05830123), aiming to delve deeper into HS-20093's efficacy and safety in this patient population. The findings from this phase 1 trial represent a significant stride towards addressing the urgent medical need for effective treatments for relapsed and refractory sarcomas, offering renewed hope to patients facing this challenging journey.

Phase 1 Results of Azenosertib plus Gemcitabine in Patients with Relapsed or Refractory Osteosarcoma

Patients with recurrent or metastatic osteosarcoma have poor outcomes. Treatments to help this vulnerable population are desperately needed. Azenosertib is a novel targeted therapy against the WEE1 kinase; it specifically causes OS cancer cells to rapidly divide without pausing to repair any damage that has occurred to the cancer cells. In doing so, it causes these cancer cells to die off because they are too damaged to survive. In early trials using mouse models and OS cancer cell lines, azenosertib was found to be effective in killing OS cells. Furthermore, by combining it with gemcitabine (a cytotoxic chemotherapy agent that can be used in the treatment of OS), the cancer-killing potential as significantly higher than either drug alone. As such, this clinical trial studied what dose and combination of azenosertib with gemcitabine was safe and well tolerated and whether it slowed the growth and spread of OS. Patients 12 years of age and older were able to enroll on the clinical trial. The study sought to clarify two points: - the highest dose of this combination that was both safe and tolerable, defined as the dose that did not cause a significant side effect in more than 1 in 3 patients (a total of 5 dose combinations were studied) - the change in tumor size and number at 18 weeks based on CT or MRI scans As of Nov 30, 2023, 31 patients had been treated. Patients ranged in age from 12 to 76 years. Patients had previously received between 1 and 9 prior lines of treatment. At the highest dose that was found to be safe and tolerable, the most frequent and notable side effects were a decrease in the platelet and white cell count. Side effects that caused a decrease in the dose combination were primarily due to a lower platelet count and gastrointestinal symptoms (e.g., throwing up or diarrhea). Of the treated patients, 39% had no growth or new tumors appear at 18 weeks (this compares to 12% at 16 weeks in most prior clinical trials). Based on this, we believe that azenosertib + gemcitabine is well tolerated, safe and slowed osteosarcoma growth better than many previous clinical trials in OS. This is important because we want to study the highest dose that was found to be safe and tolerable in a bigger study to confirm these findings and hopefully, make this drug combination available to all patients, even outside clinical trials.

Data Commons to Support New Treatments for Osteosarcoma

Despite decades of progress in curing pediatric cancer, bone tumors are among the most deadly pediatric malignancies. Of children with non-metastatic osteosarcoma, the survival rate is 69%, while cure rates for tumors that have spread to distant areas is a dismal 27%. In an era of high-resolution gene sequencing and personalized medicine, it is tragic that most children with bone tumors are treated with decades-old chemotherapy regimens. The field is ripe for progress, and the key to innovation is more and better-connected data. The Pediatric Cancer Data Commons at the University of Chicago is dedicated to lowering barriers to cancer research. The PCDC creates universal data standards for pediatric malignancies, transforms data into this new standard, and offers up those data to researchers. Over the past eight years, the PCDC has developed advanced international data dictionaries for eleven pediatric tumor types and is ingesting data from tens of thousands of children. The PCDC bone sarcoma consortium, HIBiSCus (Harmonization International Bone Sarcoma Consortium), which has representation from over 15 countries and has created a harmonized international data dictionary for both osteosarcoma and Ewing sarcoma. The HIBiSCus data commons will include clinical and genomic biomarker data from thousands of children with osteosarcoma from all over the world, harmonized to a common standard, and available to researchers through an advanced cohort discovery tool and rigorous governance for data sharing. This resource will transform research for these deadly tumors, allowing researchers unprecedented access to data and enabling discovery of mechanisms of disease and novel therapies.