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Purpose: To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. Experimental Design: Patients were recruited through social media outreach and engagement with disease-specific advocacy groups, with a focus on patients with histiocytosis, germ cell tumors (GCT), and pediatric cancers. Tumors were analyzed using the MSK-IMPACT next-generation sequencing assay with the return of results to patients and their local physicians. Whole-exome recapture was performed for female patients with GCTs to define the genomic landscape of this rare cancer subtype. Results: A total of 333 patients were enrolled, and tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quality for MSK-IMPACT testing. Eighteen patients with histiocytosis have received genomically guided therapy to date, of whom 17 (94%) have had clinical benefit with a mean treatment duration of 21.7 months (range, 6–40+). Whole-exome sequencing of ovarian GCTs identified a subset with haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%); however, 2 patients with ovarian GCTs with squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. Conclusions: Direct-to-patient outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians to guide treatment.

Purpose:To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes.Experimental Design:Patients were recruited through social media outreach and engagement with disease-specific advocacy groups, with a focus on patients with histiocytosis, germ cell tumors (GCT), and pediatric cancers. Tumors were analyzed using the MSK-IMPACT next-generation sequencing assay with the return of results to patients and their local physicians. Whole-exome recapture was performed for female patients with GCTs to define the genomic landscape of this rare cancer subtype.Results:A total of 333 patients were enrolled, and tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quality for MSK-IMPACT testing. Eighteen patients with histiocytosis have received genomically guided therapy to date, of whom 17 (94%) have had clinical benefit with a mean treatment duration of 21.7 months (range, 6–40+). Whole-exome sequencing of ovarian GCTs identified a subset with haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%); however, 2 patients with ovarian GCTs with squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab.Conclusions:Direct-to-patient outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians to guide treatment.

Genomically matched targeted therapies guided by MSK-IMPACT testing (n=22).

Clonality assessment of known oncogenic mutations in the female GCT cohort.

Gene fusions frequently result from rearrangements in cancer genomes. In many instances, gene fusions play an important role in oncogenesis; in other instances, they are thought to be passenger events. Although regulatory element rearrangements and copy number alterations resulting from these structural variants are known to lead to transcriptional dysregulation across cancers, the extent to which these events result in functional dependencies with an impact on cancer cell survival is variable. Here we used CRISPR-Cas9 dependency screens to evaluate the fitness impact of 3,277 fusions across 645 cell lines from the Cancer Dependency Map. We found that 35% of cell lines harbored either a fusion partner dependency or a collateral dependency on a gene within the same topologically associating domain as a fusion partner. Fusion-associated dependencies revealed numerous novel oncogenic drivers and clinically translatable alterations. Broadly, fusions can result in partner and collateral dependencies that have biological and clinical relevance across cancer types.Significance:This study provides insights into how fusions contribute to fitness in different cancer contexts beyond partner-gene activation events, identifying partner and collateral dependencies that may have direct implications for clinical care.

Supplementary Data from Gene Fusions Create Partner and Collateral Dependencies Essential to Cancer Cell Survival

The extent to which clinical and genomic characteristics associate with prostate cancer clonal architecture, tumor evolution, and therapeutic response remains unclear. Here, we reconstructed the clonal architecture and evolutionary trajectories of 845 prostate cancer tumors with harmonized clinical and molecular data. We observed that tumors from patients who self-reported as Black had more linear and monoclonal architectures, despite these men having higher rates of biochemical recurrence. This finding contrasts with prior observations relating polyclonal architecture to adverse clinical outcomes. Additionally, we utilized a novel approach to mutational signature analysis that leverages clonal architecture to uncover additional cases of homologous recombination and mismatch repair deficiency in primary and metastatic tumors and link the origin of mutational signatures to specific subclones. Broadly, prostate cancer clonal architecture analysis reveals novel biological insights that may be immediately clinically actionable and provide multiple opportunities for subsequent investigation.Statement of significanceTumors from patients who self-reported as Black demonstrate linear and monoclonal evolutionary trajectories yet experience higher rates of biochemical recurrence. In addition, analysis of clonal and subclonal mutational signatures identifies additional tumors with potentially actionable alterations such as deficiencies in mismatch repair and homologous recombination.

SUMMARYCells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series ofin vivomodels to induceGclcdeletion in adult animals. We find that GSH is essential to lipid abundancein vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver’s balance of redox buffering and triglyceride production.

Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.

Molecular profiling studies have enabled discoveries for metastatic prostate cancer (MPC) but have predominantly occurred in academic medical institutions and involved non-representative patient populations. We established the Metastatic Prostate Cancer Project (MPCproject, mpcproject.org), a patient-partnered initiative to involve patients with MPC living anywhere in the US and Canada in molecular research. Here, we present results from our partnership with the first 706 MPCproject participants. While 41% of patient partners live in rural, physician-shortage, or medically underserved areas, the MPCproject has not yet achieved racial diversity, a disparity that demands new initiatives detailed herein. Among molecular data from 333 patient partners (572 samples), exome sequencing of 63 tumor and 19 cell-free DNA (cfDNA) samples recapitulated known findings in MPC, while inexpensive ultra-low-coverage sequencing of 318 cfDNA samples revealed clinically relevant

Purpose: Enzalutamide is a second-generation androgen receptor (AR) inhibitor that has improved overall survival (OS) in metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients develop resistance. We designed a phase II multicenter study of enzalutamide in metastatic CRPC incorporating tissue and blood biomarkers to dissect mechanisms driving resistance. Patients and Methods: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy and then initiated enzalutamide 160 mg daily. A repeat metastasis biopsy was obtained at radiographic progression from the same site when possible. Blood for circulating tumor cell (CTC) analysis was collected at baseline and progression. The primary objective was to analyze mechanisms of resistance in serial biopsies. Whole-exome sequencing was performed on tissue biopsies. CTC samples underwent RNA sequencing. Results: A total of 65 patients initiated treatment, of whom 22 (33.8%) had received prior abiraterone. Baseline biopsies were enriched for alterations in AR (mutations, amplifications) and tumor suppression genes (PTEN, RB1, and TP53), which were observed in 73.1% and 92.3% of baseline biopsies, respectively. Progression biopsies revealed increased AR amplifications (64.7% at progression vs. 53.9% at baseline) and BRCA2 alterations (64.7% at progression vs. 38.5% at baseline). Genomic analysis of baseline and progression CTC samples demonstrated increased AR splice variants, AR-regulated genes, and neuroendocrine markers at progression. Conclusions: Our results demonstrate that a large proportion of enzalutamide-treated patients have baseline and progression alterations in the AR pathway and tumor suppressor genes. We demonstrate an increased number of BRCA2 alterations post-enzalutamide, highlighting the importance of serial tumor sampling in CRPC.

Metastatic castration-resistant prostate cancer is typically lethal, exhibiting intrinsic or acquired resistance to second-generation androgen-targeting therapies and minimal response to immune checkpoint inhibitors1. Cellular programs driving resistance in both cancer and immune cells remain poorly understood. We present single-cell transcriptomes from 14 patients with advanced prostate cancer, spanning all common metastatic sites. Irrespective of treatment exposure, adenocarcinoma cells pervasively coexpressed multiple androgen receptor isoforms, including truncated isoforms hypothesized to mediate resistance to androgen-targeting therapies2,3. Resistance to enzalutamide was associated with cancer cell–intrinsic epithelial–mesenchymal transition and transforming growth factor-β signaling. Small cell carcinoma cells exhibited divergent expression programs driven by transcriptional regulators promoting lineage plasticity and HOXB5, HOXB6 and NR1D2 (refs. 4–6). Additionally, a subset of patients had high expression of dysfunction markers on cytotoxic CD8+ T cells undergoing clonal expansion following enzalutamide treatment. Collectively, the transcriptional characterization of cancer and immune cells from human metastatic castration-resistant prostate cancer provides a basis for the development of therapeutic approaches complementing androgen signaling inhibition., Single-cell transcriptomic analysis of metastatic castration-resistant prostate cancer uncovers pervasive coexpression of androgen receptor isoforms and cancer cell–intrinsic and microenvironmental programs of treatment resistance

Although papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor with a generally excellent prognosis, a patient developed a clinically aggressive PTC eleven years after receiving platinum chemotherapy for ovarian endometrioid adenocarcinoma. Germline and somatic analyses of multi-temporal and multi-regional molecular profiles indicated that ovarian and thyroid tumors did not share common genetic alterations. PTC tumors had driver events associated with aggressive PTC behavior, an RBPMS-NTRK3 fusion and a TERT promoter mutation. Spatial and temporal genomic heterogeneity analysis indicated a close link between anatomical locations and molecular patterns of PTC. Mutational signature analyses demonstrated a molecular footprint of platinum exposure, and that aggressive molecular drivers of PTC were linked to prior platinum-associated mutagenesis. This case provides a direct association between platinum chemotherapy exposure and secondary solid tumor evolution, in specific aggressive thyroid carcinoma, and suggests that uniform clinical assessments for secondary PTC after platinum chemotherapy may warrant further evaluation.

Translational oncology studies increasingly emphasize deriving hypotheses from single-cell datasets by evaluating groups of patients (e.g., treated vs. untreated, responders vs. non-responders, early vs. late-stage tumors) for differences in gene signatures/pathways to guide subsequent investigations. However, when evaluating these questions using tumor single-cell data, current statistical methods for this task are limited in two major aspects: (i) They are not representative of the hierarchical nature of tumor single-cell datasets (where cells are more similar within the patient than between patients); and (ii) They often do not contextualize statistical significance (p-values) in the context of variability that is expected biologically between patients. This can lead to molecular hypotheses that are skewed by patient-specific biology with high false positive rates. To address these challenges, we developed a nonparametric statistical method, BEANIE (group Biology EstimAtioN in sIngle cEll), for estimating group biology in single-cell transcriptomic datasets. BEANIE uses monte carlo simulations to estimate the p-value distribution for the gene signature of interest and normalizes with respect to a background distribution of the expected patient-to-patient variability. The method then performs leave-one-out cross-validation to infer robustness of the gene signatures to patient exclusion. We applied our method to public cancer single-cell datasets to evaluate candidate differences in tumor cell populations between known groups. When comparing tumor cell programs derived from treatment-naive vs. chemotherapy-treated Triple-Negative Breast Cancer (TNBC) patients, BEANIE identified upregulation of hallmark gene signatures for fatty acid metabolism and hypoxia to be statistically significant and robust in the treatment-naive group, indicative of response to chemotherapy treatment. We also compared BEANIE to conventional methods like Mann-Whitney U (MWU) test followed by Benjamini-Hochberg (BH) correction and Generalized Linear Models (GLM). MWU test and GLM also identified these signatures to be upregulated, but they additionally identified other immune cell gene signatures like T-cell markers, B-cell markers and NK cell markers, to also be upregulated, which was not expected since we were comparing between tumor cells. Similarly, BEANIE identified candidate tumor cell intrinsic programs relevant to hypotheses in lung cancer patients (early vs. late-stage) and melanoma patients (Immune Checkpoint Blockade (ICB)-naive vs. ICB-treated). Therefore, BEANIE greatly increased the specificity of analysis, and reduced false positive rates. This method is publicly available and may overcome existing methodological limitations to guide tumor-intrinsic hypothesis generation in rapidly growing clinical cancer single-cell cohorts. Citation Format: Shreya Johri, Kevin Bi, Breanna M. Titchen, Jingxin Fu, Jake Conway, Jett Crowdis, Natalie I. Volkes, Zenghua Fan, Lawrence Fong, Jihye Park, David Liu, Meng Xiao He, Eliezer M. Van Allen. Dissecting tumor cell programs through group biology estimation in clinical single-cell transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1120.

203 Background: Radium-223 (Ra-223) is a bone-seeking alpha emitter that induces double-stranded DNA breaks, and the homologous recombination (HR) pathway is critical for repairing these breaks. While prior studies suggested that metastatic castrate-resistant prostate cancers (mCRPC) patients (pts) with HR-deficient (HRD+) tumors may be more likely to benefit from Ra-223, obtaining tissue for next generation sequencing to identify HRD+ is challenging in pts with bone-predominant disease. We hypothesized that circulating tumor DNA (ctDNA) would allow for broader identification of HRD+ to assess association with clinical outcomes in a real-world cohort. Methods: We identified 135 mCRPC pts treated with Ra-223 at our institution between 2013 and 2021. Pts who initiated another anti-tumor therapy within 60 days of Ra-223 treatment were excluded; pts continuing hormonal agents initiated >60 days prior were included. ctDNA isolated from pre-treatment plasma underwent ultra-low-pass whole genome sequencing to estimate tumor fraction (TF). Additionally, targeted panel sequencing using an institutional prostate cancer-specific panel of 319 genes with duplex sequencing (utilizing unique molecular identifiers) for error suppression was used to identify germline or somatic deleterious alterations in HR pathway genes. The primary outcome was association between HRD status and completion of fewer than 6 cycles (as a proxy for early clinical progression), assessed using logistic regression. Results: The median age was 61 (IQR, 56-67) years, median pretreatment prostate-specific antigen (PSA) level was 26.2 (IQR, 8.1-84.1) ng/mL, and median TF was 4% (IQR, 3-6%). 97% of pts (n=131) previously received a novel antiandrogen, and 63% (n=85) received prior taxane. 17% (n=23) were HRD+, and 59% (n=80) completed 6 cycles of Ra-223. On multivariable analysis, HRD+ was associated with decreased likelihood of completing 6 cycles compared to HRD- (adjusted odds ratio [AOR] 0.16, 95% confidence interval [CI] 0.05-0.48, P=0.001). 22% (n=5) of HRD+ pts completed 6 cycles compared to 67% (n=75) of HRD- pts. Additional factors associated with decreased likelihood of completing 6 cycles included a higher pretreatment TF (AOR 0.69, 95% CI, 0.48-0.97, P=0.034) and prior taxane use (AOR 0.41, 95% CI, 0.18-0.91, P=0.028), but not pretreatment PSA ( P=0.574). Conclusions: Targeted panel sequencing with error suppression from ctDNA identified HRD+ in mCRPC pts with bone-predominant disease and low median TF at a similar frequency as reported from tissue. In our cohort, HRD+ was prognostic of early clinical progression with Ra-223. Further work is in progress to understand the association of other ctDNA-derived features, including assessment of genomic signatures and transcriptional binding sites, in the setting of Ra-223 therapy. DF/HCC IRB protocol 18-135.

Molecular profiling studies have enabled numerous discoveries for metastatic prostate cancer (MPC), but they have mostly occurred in academic medical institutions focused on select patient populations. We developed the Metastatic Prostate Cancer Project (MPCproject, mpcproject.org), a patient-partnered initiative to empower MPC patients living anywhere in the U.S. and Canada to participate in molecular research and contribute directly to translational discovery. Here we present clinicogenomic results from our partnership with the first 706 MPCproject participants. We found that a patient-centered and remote research strategy enhanced engagement with patients in rural and medically underserved areas. Furthermore, patient-reported data achieved 90% consistency with abstracted health records for therapies and provided a mechanism for patient-partners to share information about their cancer experience not documented in medical records. Among the molecular profiling data from 333 patient-partners (n = 573 samples), whole exome sequencing of 63 tumor samples obtained from hospitals across the U.S. and Canada and 19 plasma cell-free DNA (cfDNA) samples from blood donated remotely recapitulated known findings in MPC and enabled longitudinal study of prostate cancer evolution. Inexpensive ultra-low coverage whole genome sequencing of 318 cfDNA samples from donated blood revealed clinically relevant genomic changes like AR amplification, even in the context of low tumor burden. Collectively, this study illustrates the power of a longitudinal partnership with patients to generate a more representative clinical and molecular understanding of MPC.NoteTo assist our patient-partners and the wider MPC community interpret the results of this study, we have included a glossary of terms in the Supplementary Materials.

Gene fusions frequently result from rearrangements in cancer genomes. In many instances, gene fusions play an important role in oncogenesis; in other instances, they are thought to be passenger events. Although regulatory element rearrangements and copy number alterations resulting from these structural variants are known to lead to transcriptional dysregulation across cancers, the extent to which these events result in functional dependencies with an impact on cancer cell survival is variable. Here we used CRISPR-Cas9 dependency screens to evaluate the fitness impact of 3,277 fusions across 645 cell lines from the Cancer Dependency Map. We found that 35% of cell lines harbored either a fusion partner dependency or a collateral dependency on a gene within the same topologically associating domain as a fusion partner. Fusion-associated dependencies revealed numerous novel oncogenic drivers and clinically translatable alterations. Broadly, fusions can result in partner and collateral dependencies that have biological and clinical relevance across cancer types. Significance: This study provides insights into how fusions contribute to fitness in different cancer contexts beyond partner-gene activation events, identifying partner and collateral dependencies that may have direct implications for clinical care.

6532 Background: Tumor genomic profiling is increasingly used to identify actionable genomic alterations as a guide to therapy selection. To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. Methods: Patients were recruited through social media outreach, engagement with disease advocacy groups, or via physician referral, with a focus on recruiting patients with histiocytosis, germ cell tumors and rare pediatric cancers. Tumor and patient-matched germline DNA were analyzed using the MSK-IMPACT targeted sequencing next generation sequencing panel with return of results to patients and their local physicians. Whole exome recapture of MSK-IMPACT DNA sequencing libraries was performed for patients with female germ cell tumors to define the genomic landscape of this rare cancer subtype. Results: 359 cancer patients expressed interest in the Make-an-IMPACT program, of whom 333 were enrolled. Tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quantity and quality for MSK-IMPACT testing. 14 histiocytosis patients have received genomically guided therapy to date, of whom 13 (93%) have had clinical benefit based on local MD response assessment with a mean treatment duration of 16.7 months (range 3-32+). Whole exome sequencing of ovarian GCTs identified a subset with fully haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%), however, 2 ovarian GCTs and squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. Conclusions: Social media outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians where they could be used to guide treatment selection. This can also open the door to diversifying and being able to study the genomic landscape in a diverse cohort.

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyzed cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single cell analysis of human clinical samples exhibit a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

Metastatic castration resistant prostate cancer (mCRPC) is primarily treated with therapies that prevent transcriptional activity of the androgen receptor (AR), cause DNA damage, or prevent cell division. Clinical resistance to these therapies, including second-generation androgen-targeting compounds such as enzalutamide and abiraterone, is nearly universal. Other treatment modalities, including immune checkpoint inhibitors, have provided minimal benefit except in rare subsets of patients1,2. Both tumour intrinsic and extrinsic cellular programs contributing to therapeutic resistance remain areas of active investigation. Here we use full-length single-cell RNA-sequencing (scRNA-seq) to identify the transcriptional states of cancer and immune cells in the mCRPC microenvironment. Within cancer cells, we identified transcriptional patterns that mediate a significant proportion of inherited risk for prostate cancer, extensive heterogeneity inARsplicing within and between tumours, and vastly divergent regulatory programs between adenocarcinoma and small cell carcinoma. Moreover, upregulation of TGF-β signalling and epithelial-mesenchymal transition (EMT) were both associated with resistance to enzalutamide. We found that some lymph node metastases, but no bone metastases, were heavily infiltrated by dysfunctional CD8+T cells, including cells undergoing dramatic clonal expansion during enzalutamide treatment. Our findings suggest avenues for rational therapeutic approaches targeting both tumour-intrinsic and immunological pathways to combat resistance to current treatment options.

High-risk localized prostate cancer (HRLPC) is associated with a substantial risk of recurrence and prostate cancer-specific mortality1. Recent clinical trials have shown that intensifying anti-androgen therapies administered prior to prostatectomy can induce pathologic complete responses (pCR) or minimal residual disease (MRD) (SPOP mutation and SPOPL copy number loss were exclusively observed in ER, while TP53 mutation and PTEN copy number loss were exclusively observed in NR. These alterations were clonal in all tumor phylogenies per patient. Additionally, transcriptional programs involving androgen signaling and TGFβ signaling were enriched in ER and NR, respectively. The presence of these alterations in routine biopsies from patients with HRLPC may inform the prospective identification of responders to neoadjuvant anti-androgen therapies to improve clinical outcomes and stratify other patients to alternative biologically informed treatment strategies.

MotivationLarge-scale sequencing studies have created a need to succinctly visualize genomic characteristics of patient cohorts linked to widely variable phenotypic information. This is often done by visualizing the co-occurrence of variants with comutation plots. Current tools lack the ability to create highly customizable and publication quality comutation plots from arbitrary user data.ResultsWe developed CoMut, a stand-alone, object-oriented Python package that creates comutation plots from arbitrary input data, including categorical data, continuous data, bar graphs, side bar graphs, and data that describes relationships between samples.Availability and ImplementationThe CoMut package is open source and is available at https://github.com/vanallenlab/comut under the MIT License, along with documentation and examples. A no installation, easy-to-use implementation is available on Google Colab (see GitHub).Contacteliezerm_vanallen@dfci.harvard.edu

Gene fusions frequently result from rearrangements in cancer genomes. In many instances, they play an important role in oncogenesis; in other instances, they are thought to be passenger events. Regulatory element rearrangements and copy number alterations resulting from these structural variants (SVs) lead to transcriptional dysregulation across cancers, though the extent to which these events result in functional dependencies with an impact on cancer cell survival is variable. We demonstrated enrichment of fusion partner genes and “collateral genes”, or genes in the same topologically associating domains (TADs) as fusion partners, among CRISPR-Cas9 dependencies across 209 cell lines with whole-genome sequencing data. We subsequently used CRISPR-Cas9 dependency screens to evaluate the fitness impact of 3,277 fusions across 645 cell lines with RNAseq data from the Cancer Dependency Map (DepMap). We found that 35% of cell lines harbored either a fusion partner dependency or a collateral dependency. CRISPR-Cas9 loss-of-function screening identified fusion-associated dependencies in 20 of 35 (57%) COSMIC fusions from our dataset. Fusion-associated dependencies revealed numerous novel oncogenic drivers and clinically translatable alterations. We observed several instances of intrachromosomal FOXR1 fusions associated with FOXR1 as a strong dependency, occurring independently in osteosarcoma, lung adenocarcinoma, and bladder carcinoma cell lines; these dependencies were validated through in vitro CRISPR-Cas9 knockout. FOXR1 fusions leading to FOXR1 overexpression were seen in an independent cohort of clinical tumor samples, speaking to the clinical relevance of this finding. FGFR3, a targetable kinase, was the key dependency in t(4;14) multiple myeloma cells that harbored an IgH-NSD2 fusion. We also showed that the implications of fusion-associated dependencies extended beyond two-dimensional cell line space, exemplified by dependency on EML4 in the context of a THADA-MTA3 fusion persisting in spheroid models. Broadly, fusions can result in partner and collateral dependencies that have biological and clinical relevance across cancer types. Citation Format: Riaz Gillani, Bo Kyung A. Seong, Jett Crowdis, Jake R. Conway, Neekesh V. Dharia, Saif Alimohamed, Brian J. Haas, Kyuho Han, Jihye Park, David Liu, Felix Dietlein, Meng Xiao He, Alma Imamovic, Clement Ma, Michael C. Bassik, Jesse S. Boehm, Francisca Vazquez, Alexander Gusev, Katherine A. Janeway, James M. McFarland, Kimberly Stegmaier, Eliezer M. Van Allen. Gene fusions create partner and collateral dependencies that are essential to cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 96.

Poison frogs acquire chemical defenses from the environment for protection against potential predators. These defensive chemicals are lipophilic alkaloid toxins that are sequestered by poison frogs from dietary arthropods and stored in skin glands. Despite decades of research focusing on identifying poison frog toxins, we know relatively little about how environmental variation and subsequent arthropod availability impacts toxicity in poison frogs. We investigated how seasonal environmental variation influences poison frog toxin profiles through changes in the diet of the Climbing Mantella (Mantella laevigata). We collected M. laevigata females on the Nosy Mangabe island reserve in Madagascar during the wet and dry seasons and tested the hypothesis that seasonal differences in rainfall is associated with changes in the diet and skin toxin profiles of M. laevigata. The arthropod diet of each frog was characterized into five groups (i.e. ants, termites, mites, insect larvae, or ‘other’) using visual identification and cytochrome oxidase 1 DNA barcoding. We found that frog diet differed between the wet and dry seasons, where frogs had a more diverse diet in the wet season and consumed a higher percentage of ants in the dry season. To determine if seasonality was associated with variation in frog defensive chemical composition, we used gas chromatography / mass spectrometry to quantify toxins from individual skin samples. Although the assortment of identified toxins was similar across seasons, we detected significant differences in the abundance of certain alkaloids, which we hypothesize reflects seasonal variation in the diet of M. laevigata. We suggest that these variations could originate from seasonal changes in either arthropod leaf litter composition or changes in frog behavioral patterns. Although additional studies are needed to understand the consequences of long-term environmental shifts, this work suggests that toxin profiles are relatively robust against short-term environmental perturbations.

Poison frogs acquire chemical defenses from the environment for protection against potential predators. These defensive chemicals are lipophilic alkaloids that are sequestered by poison frogs from dietary arthropods and stored in skin glands. Despite decades of research focusing on identifying poison frog alkaloids, we know relatively little about how environmental variation and subsequent arthropod availability impacts alkaloid loads in poison frogs. We investigated how seasonal environmental variation influences poison frog chemical profiles through changes in the diet of the Climbing Mantella (Mantella laevigata). We collected M. laevigata females on the Nosy Mangabe island reserve in Madagascar during the wet and dry seasons and tested the hypothesis that seasonal differences in rainfall is associated with changes in diet composition and skin alkaloid profiles of M. laevigata. The arthropod diet of each frog was characterized into five groups (i.e. ants, termites, mites, insect larvae, or 'other') using visual identification and cytochrome oxidase 1 DNA barcoding. We found that frog diet differed between the wet and dry seasons, where frogs had a more diverse diet in the wet season and consumed a higher percentage of ants in the dry season. To determine if seasonality was associated with variation in frog defensive chemical composition, we used gas chromatography / mass spectrometry to quantify alkaloids from individual skin samples. Although the assortment of identified alkaloids was similar across seasons, we detected significant differences in the abundance of certain alkaloids, which we hypothesize reflects seasonal variation in the diet of M. laevigata. We suggest that these variations could originate from seasonal changes in either arthropod leaf litter composition or changes in frog behavioral patterns. Although additional studies are needed to understand the consequences of long-term environmental shifts, this work suggests that alkaloid profiles are relatively robust against short-term environmental perturbations.

Tumors evolve through progressive accumulation of (epi)genetic alterations, favoring expansion of the fittest cell populations as a result of stimuli and selective pressures in the microenvironment. Intratumor heterogeneity and the interplay of tumor cells with the microenvironment present an order of complexity that can have profound effects on tumor progression and drug sensitivity. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of functional heterogeneity within a single tumor, and the dynamics and spatial organization of distinct cell populations over time. The overall objective of this study is to characterize the functional heterogeneity of breast tumor cells and evaluate the dynamics of clonal populations within mixtures during tumor progression. We have generated and characterized 31 clonal cell populations (SCP) from the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic heterogeneity with respect to morphology, proliferation rate, survival in suspension, colony formation in soft agar, and tumorigenicity. We have also performed whole-exome sequencing for 7 SCPs, and RNA-seq and RPPA analysis for all SCPs and the parental cell line. These analyses revealed SCP-unique single nucleotide variants, and differential expression of numerous genes. To study the dynamics of SCP mixtures during the development of xenograft tumors, we have transduced 22 SCPs with unique DNA barcodes derived from the ClonTracer DNA barcode library. The 22 barcoded SCPs were mixed in equal proportions and subsequently injected into the mammary fat pad of NOD/SCID mice. Tumors and lungs were collected at different time points (3 weeks, 2 months and 4 months) to determine the relative changes in clonal representation during tumor progression by next generation sequencing. We identified highly reproducible patterns of clonal expansion, with SCP01 and SCP03 being temporarily enriched for in tumor samples collected at 3 weeks and 2 months respectively, and SCP32 becoming progressively enriched for in tumors over time. In addition, we found recurrent enrichment for SCP01 in most lung samples. Moreover, in contrast to the tumor samples, we found a slight enrichment for SCP13, but not SCP32, during in vitro propagation of the SCP mixture. Together these results suggest the existence of distinct competitive advantages of individual clonal populations within certain spatial and temporal windows. In addition, the discordance in SCP dynamics between the in vivo and in vitro arms suggests that enrichment for SCP01, SCP03 and SCP32 is the result of stimuli or selective pressures that are specific to the tumor microenvironment. Guided by the RNA-seq and RPPA analyses, we are currently testing several hypotheses that may explain the observed enrichment patterns. Citation Format: Hendrik J. Kuiken, Sabin Dhakal, Laura M. Selfors, Jett P. Crowdis, Hyo-eun C. Bhang, Frank Stegmeier, Gordon B. Mills, Joan S. Brugge. Characterization of functional heterogeneity and in vivo dynamics of clonal cell populations derived from the triple-negative breast cancer cell line MDA-MB-468 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2017-3957