Focal Copy Number Research Articles

Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia

AbstractOncogenes can be activated in cis through multiple mechanisms including enhancer hijacking events and noncoding mutations that create enhancers or promoters de novo. These paradigms have helped parse somatic variation of noncoding cancer genomes, thereby providing a rationale to identify noncanonical mechanisms of gene activation. Here we describe a novel mechanism of oncogene activation whereby focal copy number loss of an intronic element within the FTO gene leads to aberrant expression of IRX3, an oncogene in T-cell acute lymphoblastic leukemia. Loss of this CTCF-bound element downstream to IRX3 (+224 kb) leads to enhancer hijack of an upstream developmentally active super-enhancer of the CRNDE long noncoding RNA (−644 kb). Unexpectedly, the CRNDE super-enhancer interacts with the IRX3 promoter with no transcriptional output until it is untethered from the FTO intronic site. We propose that “promoter tethering” of oncogenes to inert regions of the genome is a previously unappreciated biological mechanism preventing tumorigenesis.

Whole-exome sequencing reveals novel cancer genes and actionable targets in biliary tract cancers in primary sclerosing cholangitis.

People with primary sclerosing cholangitis (PSC) have a 20% lifetime risk of biliary tract cancer (BTC). Using whole-exome sequencing, we characterized genomic alterations in tissue samples from BTC with underlying PSC. We extracted DNA from formalin-fixed, paraffin-embedded tumor and paired nontumor tissue from 52 resection or biopsy specimens from patients with PSC and BTC and performed whole-exome sequencing. Following copy number analysis, variant calling, and filtering, putative PSC-BTC-associated genes were assessed by pathway analyses and annotated to targeted cancer therapies. We identified 53 candidate cancer genes with a total of 123 nonsynonymous alterations passing filtering thresholds in 2 or more samples. Of the identified genes, 19% had not previously been implicated in BTC, including CNGA3, KRT28, and EFCAB5. Another subset comprised genes previously implicated in hepato-pancreato-biliary cancer, such as ARID2, ELF3, and PTPRD. Finally, we identified a subset of genes implicated in a wide range of cancers such as the tumor suppressor genes TP53, CDKN2A, SMAD4, and RNF43 and the oncogenes KRAS, ERBB2, and BRAF. Focal copy number variations were found in 51.9% of the samples. Alterations in potential actionable genes, including ERBB2, MDM2, and FGFR3 were identified and alterations in the RTK/RAS (p = 0.036), TP53 (p = 0.04), and PI3K (p = 0.043) pathways were significantly associated with reduced overall survival. In this exome-wide characterization of PSC-associated BTC, we delineated both PSC-specific and universal cancer genes. Our findings provide opportunities for a better understanding of the development of BTC in PSC and could be used as a platform to develop personalized treatment approaches.

Abstract 4346: Deciphering the landscape of extrachromosomal DNA in gastric cancer and its potential impact on cancer dynamics

Abstract Extrachromosomal DNA (ecDNA) drives substantial copy number increases in focal regions harboring oncogenes and its non-Mendelian inheritance during mitosis contributes to cancer heterogeneity and a poor prognosis across multiple cancer types. However, the prevalence and function of ecDNA in gastric cancer (GC) are still largely unknown. Here, we profiled ecDNA by whole-genome sequencing analysis from 76 GC patients and explored its functional attributes through whole-transcriptome sequencing from the same tissues. Focal somatic copy number amplification (fSCNA) was detected in 27 patients (35.5%), of which 17 (22.4%) had at least one circular form of focal amplification (ecDNA+/fSCNA+). Most of the patients carrying one or more ecDNA were classified as TCGA-CIN subtype (n=12; CIN: chromosomal instability). The fSCNA hotspots were observed at 17q12 and 19q12, where ERBB2 and CCNE1 are located, respectively. Amplifications at both loci occurred in seven patients and were formed into ecDNA in four and two patients, respectively. The ecDNA amplicons carried more oncogenes with significantly higher copy numbers than other amplicons (one-tailed Mann-Whitney U test, p=3.9e-03), and 44.4% (12 of 27) of ecDNA amplicons regions were overlapped with tandem duplication or chromothripsis, known causes of ecDNA formation. Oncogenes such as ERBB2 and MIEN1, residing on ecDNA, exhibited significantly increased expression compared to matched normal tissue as well as patients with the corresponding genes located on their chromosome only, suggesting that the ecDNA-induced copy number increases contribute to elevated expression levels. On the other hand, MYC showed higher expression in the ecDNA+/fSCNA+ group, irrespective of its fSCNA status (padj=0.028 and 4.24e-06 against the ecDNA-/fSCNA+ and ecDNA-/fSCNA- group, respectively). We further explored gene clusters that were associated with the ecDNA+/fSCNA+ group using weighted gene co-expression network (WGCNA) analysis. One module was distinct from ecDNA- patients, consisting of genes associated with the “protein processing in endoplasmic reticulum” and “spliceosome” pathways, inferring that transcription and translation are more active in ecDNA+ patients. Moreover, the overall survival analysis of 76 GC patients revealed that ecDNA+/fSCNA+ patients were significantly associated with poor prognosis (log-rank test, p=0.014). In conclusion, ecDNA may be a main source of high expression of oncogenes that influence GC development, and subsequently upregulated global transcription activity will make GC more aggressive. Citation Format: Donghyeok Seol, Seunghyun Kang, Sanghyun Kim, Jieun Lee, Mira Yoo, Duyeong Hwang, So Hyun Kang, Chanmi Bang, Young Suk Park, Sang-Hoon Ahn, Hyung-Ho Kim, Hoon Kim, Yun-Suhk Suh. Deciphering the landscape of extrachromosomal DNA in gastric cancer and its potential impact on cancer dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4346.

Abstract 3931: Breast cancer genomic architecture contributes to immune escape across metastasis

Abstract Clinically, breast tumors are stratified based on the expression of three receptors - estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). Breast cancer is a highly heterogeneous disease, and a more detailed understanding of its genomic drivers, mechanisms of immune evasion, and evolution may inform more effective and personalized treatment. Previously, we established a genome-driven breast cancer classification scheme that defines 11 integrative subgroups (ICs) of disease with distinct copy number aberrations (CNAs), transcriptional profiles, and clinical outcomes. Specifically, we identify four subgroups of ER+ disease (IC1, IC2, IC6, IC9) with a persistent risk of lethal distant relapse up to two decades after diagnosis, each with focal copy number drivers and two distinct subgroups of triple-negative disease (Curtis et al. Nature 2012; Rueda et al. Nature 2019). These findings nominate new therapeutic strategies, however, it is not known how mutational processes and genomic architecture differ across the ICs to sculpt the development and evolution of disease, nor how their microenvironments differ. To interrogate the genomic and immune landscape of breast cancer throughout the disease continuum, we established a meta-cohort of 2,873 breast tumors spanning pre-invasive (ductal carcinoma in situ), primary invasive, and metastatic breast cancer, including 1,865 with whole-genome sequencing, 1,738 with whole-transcriptome sequencing, and 730 with both modalities. We developed a fully containerized bioinformatic workflow to interrogate copy number alterations, complex classes of structural variation, and neoantigens. Analysis of this large clinically curated compendium identifies highly concordant mutational signatures and higher order genomic features in high-risk ER+ (IC1,2,6,9) and HER2+ (IC5) tumors, including distinct patterns of structural variation. In contrast, triple-negative tumors (predominantly IC10) were characterized by global genomic instability and tandem duplications, whilst a third group is largely genomically stable. These distinct genomic architectures were associated with specific tumor microenvironments. As expected, triple-negative tumors were immune-enriched. In contrast high-risk ER+ and HER2+ tumors demonstrated immune-depleted profiles, as compared to typical risk ER+ tumors. These microenvironment classifications were maintained from primary tumors to metastatic lesions, except triple-negative tumors that exhibited increased immune depletion during metastasis. Taken together, our data demonstrate that complex structural alterations established early in breast cancer contribute to immune evasion, and persist throughout the life history of the tumor through metastasis. Citation Format: Lise Mangiante, Kathleen Houlahan, Cristina Sotomayor Vivas, Alvina Adimoelja, Seongyeol Park, Aziz Khan, Sophie Pribus, Zhicheng Ma, Jennifer Caswell-Jin, Christina Curtis. Breast cancer genomic architecture contributes to immune escape across metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3931.

Abstract IA002: Management of newly diagnosed endometrial cancers and molecular classification

Abstract In the past 50 years, the treatment of this cancer has evolved from a regimen of preoperative radiation therapy followed by hysterectomy, to individualized management consisting of surgical treatment with or without additional therapy depending on whether various risk factors are identified. After the diagnosis of endometrial carcinoma has been histologically confirmed, uterine and extrauterine risk factors affect the probability of retroperitoneal lymph node involvement and subsequent survival. Surgical staging is a critical component for the proper diagnosis and management of all endometrial carcinomas. FIGO stage is the most powerful predictor of prognosis. Surgical staging is feasible by minimally invasive techniques. Patients with significant medical comorbidities who are not acceptable candidates for surgery may be managed by alternative means. Women with EC are diagnosed under the age of 40 may be eligibile for nonoperative management if there is no clear evidence of myometrial invasion. As the contemporary management of EC continues to evolve, and the indications for lymph node dissection remain controversial. Minimally invasive management of EC has increasingly been integrated into standard practice. In 2006, The Cancer Genome Atlas (TCGA) began with studies in glioblastoma and high-grade serous ovarian cancer. The National Institutes of Health committed major resources to TCGA to collect and characterize the genomic landscape of more than 20 cancer types. In 2013, TCGA published findings for endometrial cancers. Only serous and endometrioid subtypes were accrued in the endometrial cancer project. TCGA has reclassified endometrioid and serous endometrial cancers in four broad categories using comprehensive genomic approaches. One-third of the cancers is characterized by microsatellite instability (MSI) and associated hyper-mutation. The cancers without MSI can be further separated into two groups. Serous cases with extensive DNA copy number alterations and genomic instability comprise one group, and endometrioid cases with few or only focal copy number alterations comprise the other. A smaller fourth sub-group contains samples with very high mutation rates and is characterized by mutations in POLE. Molecular features can be distinctly overlaid onto these four sub-classifications, and each have important clinical implications that are currently being used in both clinical practice and clinical trial design. In this educational presentation, we will discuss the management of newly diagnosed endometrial cancers, review the molecular classification of endometrial cancers, and identify approaches that incorporate these findings into clinical care and clinical trial design. Citation Format: Douglas A. Levine. Management of newly diagnosed endometrial cancers and molecular classification [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA002.

Development of a rapid and comprehensive genomic profiling test supporting diagnosis and research for gliomas.

A prompt and reliable molecular diagnosis for brain tumors has become crucial in precision medicine. While Comprehensive Genomic Profiling (CGP) has become feasible, there remains room for enhancement in brain tumor diagnosis due to the partial lack of essential genes and limitations in broad copy number analysis. In addition, the long turnaround time of commercially available CGPs poses an additional obstacle to the timely implementation of results in clinics. To address these challenges, we developed a CGP encompassing 113 genes, genome-wide copy number changes, and MGMT promoter methylation. Our CGP incorporates not only diagnostic genes but also supplementary genes valuable for research. Our CGP enables us to simultaneous identification of mutations, gene fusions, focal and broad copy number alterations, and MGMT promoter methylation status, with results delivered within a minimum of 4days. Validation of our CGP, through comparisons with whole-genome sequencing, RNA sequencing, and pyrosequencing, has certified its accuracy and reliability. We applied our CGP for 23 consecutive cases of intracranial mass lesions, which demonstrated its efficacy in aiding diagnosis and prognostication. Our CGP offers a comprehensive and rapid molecular profiling for gliomas, which could potentially apply to clinical practices and research primarily in the field of brain tumors.

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B092.

Abstract B082: A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC)

Abstract Introduction: KRASG12C inhibitors are a new therapeutic approach to treat KRAS mutant-driven cancers, but as with other oncogene directed therapies, adaptive resistance develops quickly and frequently. The predominant resistance mechanisms are 1) secondary mutations in KRAS and/or other mitogen-activated protein kinase (MAPK) signaling genes and 2) amplification of KRAS or MAPK genes. Extrachromosomal DNA (ecDNA) are the predominant site of focal high copy number oncogene or resistance gene amplifications in human cancers. Previous studies have demonstrated KRASG12C amplification on ecDNA to be an acquired resistance mechanism after treatment with approved KRASG12C inhibitors. Cancers enabled by ecDNA remain underserved by current therapeutic options, including targeted therapies, and have poor prognosis. An in silico “loss of function” screen revealed ribonucleotide reductase (RNR) as a critical target required for the survival of ecDNA amplified tumor cells. RNR is the rate-limiting enzyme in the biosynthesis of deoxyribonucleotide triphosphates (dNTP), the building blocks of DNA and ecDNA replication and repair. While non-selective drugs with RNR inhibitory activity (e.g., gemcitabine) indirectly validate RNR as a cancer target, no selective small molecules targeting RNR have been approved. Methods: We have developed BBI-825, a potent, selective, and orally bioavailable RNR inhibitor (RNRi) for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers. Results: BBI-825 inhibits RNR with an IC50 of 20 nM in vitro and reduces deoxyadenosine triphosphate (dATP) levels in ecDNA amplified cancer cells, with an EC50 of less than 100 nM. On target selectivity of BBI-825 was confirmed using SAFETYscan cellular screening assay investigating target selectivity over 78 targets spanning hormone receptors, kinases, calcium, GPCR, ion channel, and transporter signaling. Importantly, BBI-825 prevented resistance-conferring ecDNA-enabled amplification of KRASG12C in KRASG12C mutated colorectal cancer cells treated with the KRAS inhibitor adagrasib in vitro. Prevention of ecDNA amplification correlated with antiproliferation of BBI-825-treated tumor cells in long-term cultures. Treatment of KRASG12C mutated CRC tumor xenograft models with single agent adagrasib resulted in rapid acquired resistance through KRASG12C amplification on ecDNA. In concordance with in vitro observations, co-treatment with BBI-825 delayed the development of adagrasib resistance concurrent with an abrogation of ecDNA-enabled KRASG12C amplification (P=0.001). Furthermore, BBI-825 significantly inhibited the growth of pre-established adagrasib-resistant KRASG12C ecDNA-amplified tumors in vivo (tumor growth inhibition (TGI) = 92%, p<0.002), reinforcing clinical actionability in the KRAS inhibitor-refractory setting. Conclusions: These findings support the advancement of BBI-825 as a selective RNR inhibitor for the treatment of ecDNA-enabled oncogene or resistance gene amplified cancers, including KRASG12C mutant CRC. Citation Format: Sudhir Chowdhry, Ryan Hansen, Jacques Mauger, AnneMarie Pferdekamper, Edison Tse, Ben Norman, Jafar Moininazeri, Salvador Garcia, Yen Truong, Debbie Liao, Joshua Lange, Evan Holmes, Anthony Celeste, Dave Solis, Auzon Steffy, Joshua Plum, Julie Wiese, Julius Apuy, Ardalan Ardeshiri, Snezana Milutinovic, Deepti Wilkinson, Anthony Pinkerton, Christian Hassig, Shailaja Kasibhatla. A novel, potent and selective ribonucleotide reductase (RNR) inhibitor, BBI-825, blocks extrachromosomal DNA (ecDNA) amplification-mediated resistance to KRASG12C inhibitor in colorectal cancer (CRC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B082.

Cancer Genomic Profiling and Minimal Residual Disease Monitoring By Cell-Free DNA Sequencing in Pediatric Leukemia

Introduction: Cell-free DNA (cfDNA) sequencing has been reported as a promising non-invasive tool for detecting cancer and monitoring treatment response. We sought to evaluate the potential of a cfDNA-based strategy for detecting diverse classes of somatic mutations relevant to pediatric leukemia and cancers. Methods: Plasma samples collected at diagnosis from 135 acute lymphoblastic leukemia (ALL) and 34 acute myeloid leukemia (AML) patients were evaluated, including 114 B-ALL and 21 T-ALL. We also studied diagnostic plasma samples from 38 solid tumor and 19 brain tumor patients, and plasma collected from 12 patients with B-ALL at days 15, 22 and end of induction chemotherapy (EOI). Plasma cfDNA extraction and library preparation were performed with the NucleoSnap cfDNA kit and ThruPLEX Tag-Seq HV kit, respectively. Next-generation sequencing was performed using a capture panel covering 355 pediatric cancer genes (including noncoding regions covering hotspots of structural variation) and 8,103 heterozygous population polymorphisms. We developed analytic approaches for de novo detection of all types of somatic mutations, including focal copy number alterations, rearrangements, and large chromosomal abnormalities. Whole-genome sequencing (WGS) was also performed for 10 cfDNA samples from leukemia. Data were compared with corresponding whole-genome, exome and transcriptome tumor cell sequencing data at diagnosis. Results: At diagnosis, plasma cfDNA yield was higher for leukemias than for solid and brain tumors (median, 142 ng/ml, 7.16 ng/ml, and 7.07 ng/ml, respectively. The average coverages of leukemia cfDNA sequencing were 790X by capture panel post error suppression and 69X by WGS. We found that 100% cases of leukemia at diagnosis had detectable circulating tumor DNA (ctDNA) in plasma cfDNA, and the median ctDNA purity was 77%, comparable to primary tumor purity from diagnostic sequencing. Overall, our panel covered 86% somatic variants present in the tumor samples. Of those covered events, capture-based cfDNA sequencing detected 98% SNVs/indels, 95% focal copy gains/losses, 100% internal tandem duplications, 95% chromosomal translocations, and 99% aneuploidy. In addition, capture-based sequencing identified cfDNA-specific variants such as NRAS/KRAS SNVs and a IKZF1 deletion that were subclonal in plasma cfDNA. Of those uncovered events, 85% were structural variants with their breakpoints in intronic/noncoding regions, and cfDNA WGS identified all somatic events present in the 10 sequenced tumor samples. To detect minimal residual disease (MRD) in B-ALL patients during induction, we increased cfDNA sequencing depth for low VAF events, and the detection limit was 0.4% for SNV/indel and structural variations. Compared to the MRD results from flow cytometry, somatic mutations were detected in 7/8 MRD-positive and 1/4 MRD-negative patients on day 15, 3/4 MRD-positive patients on day 22, and 0/2 MRD-positive patients at EOI. We also sequenced cfDNA from peripheral blood and bone marrow plasma for 2 B-ALL patients on day 0, day 15, and EOI, with comparable ctDNA purity and dynamics observed between the two compartments. In a patient with concurrent ALL and neuroblastoma (NBL) who experienced NBL relapse four months after the primary diagnosis, cfDNA genomic profiling identified ctDNA derived from ALL (52% of total plasma cfDNA), primary NBL (20%), and relapsed NBL (10%), suggesting that genetic variations of multiple tumors could be detected in a single cfDNA sample. Conclusion: Our study has demonstrated the utility of cfDNA sequencing to identify both sequence and structural somatic mutations in childhood acute leukemia. This approach will be useful to monitor disease, as well as providing a non-invasive diagnostic approach.

The Impact of Soluble BCMA and BCMA Gain on Anti-BCMA Immunotherapies in Multiple Myeloma

B cell maturation antigen (BCMA) targeting chimeric antigen receptor T cells (CAR T) and bispecific T cell engagers (TCE) have remarkable efficacy in multiple myeloma (MM). While BCMA antigen escape, driven by TNFRSF17 deletions or mutations, is a mediator of anti-BCMA CAR T/ TCE resistance, the majority of patients retain BCMA surface expression at progression. Furthermore, patients with high disease burden and extramedullary disease associated with poorer response to anti-BCMA therapies, have high levels of soluble BCMA (sBCMA). The mechanisms mediating this resistance, and in particular the contribution of sBCMA, have not been fully elucidated. In order to assess the effect of sBCMA on anti-BCMA CAR T function, we measured CAR T cells activation with an in-house manufactured anti-BCMA CAR transduced with an NFAT-GFP reporter gene after co-culture with increasing concentrations of sBCMA (0-2500 ng/mL). sBCMA bound to CAR scFv in a dose dependent manner, effectively competing with the fluorophore tagged BCMA peptide binding. Despite their stable engagement, sBCMA failed to induce the expression of GFP, CD69, CD25, or 41BB on CAR T cells. To further examine the functional effect of sBCMA on CAR T cytotoxicity, we generated isogenic OPM2 cell lines with distinct BCMA expression profiles: OPM2 over-expressing BCMA (OPM2_BCMA high) generated by lentiviral transduction of TNFRSF17, and Cas9 BCMA knockout OPM2 (OPM2_BCMA -/-). OPM2_BCMA high cells exhibited one-log-fold increase in surface BCMA expression and 10-fold increase in sBCMA and were more resistant to CAR T mediated lysis. In addition, in co-culture assays, elevated sBCMA impaired OPM2 target cell lysis by anti-BCMA CAR T in a dose dependent-manner. To gain insight into the effect of chronic sBCMA exposure on CAR T, we exposed anti-BCMA CAR T to OPM2 cells secretome in the absence of direct cell to cell contact in a trans-well co-culture system. On day 24, CAR T in co-culture with OPM2_BCMA high (sBCMA levels of 2343 ng/mL) significantly contracted with no viable cells, while CAR T in culture with parental OPM2 (sBCMA level of 267 ng/mL) did persist with up-regulated TIM3 and TIGIT. In contrast CAR T cultured with OPM2_BCMA -/- remained viable with no expression of exhaustion markers. In examining the impact of sBCMA on TCE activity, sBCMA level as low as 50 ng/mL in vitro impaired anti-BCMA TCE binding to K562 cells transduced to stably express BCMA (K562_wtBCMA) . Consistent results were found in cytotoxicity assays wherein the addition of 2 ng/mL of sBCMA was sufficient to induce a reduction in anti-BCMA TCE mediated cytotoxicity of K562_wtBCMA cells by healthy donor peripheral blood mononuclear cells. To identify genomic events that lead to elevated sBCMA expression and subsequent resistance to CAR T/TCE, we conducted bulk whole genome sequencing (100X) on CD138+ bone marrow MM cells obtained from 40 MM patients treated with anti-BCMA CAR T/ TCE. Structural variants (SVs) at or adjacent to TNFRSF17 locus were identified in four patients. These included i) focal copy number gain of TNFRSF17 (3 copies) in a patient who progressed within 3 months post anti-BCMA TCE, ii) subclonal duplication of TNFRSF17 locus in another patient (n=1), as well as iii) focal copy number losses 5‘ or 3‘ to the TNFRSF17 gene locus (n=2). In addition to SVs affecting TNFRSF17 locus, we also identified mutations involving TNFRSF17 transcriptional regulators. Notably, MM clones at progression 4 months post anti-BCMA TCE (n=1) harboured copy number gains (3 copies) of POU2AF1 gene on chr11q23. POU2AF1 encodes a key transcriptional regulator of TNFRSF17 expression. Indeed, sBCMA level from this patient was significantly elevated at relapse at a level (638 ng/mL) correlated with resistance to anti-BCMA TCE. In addition, we identified translocation involving MAP3K14 encoding the NF-kB regulator NIK in a patient with short remission post anti-BCMA CAR T. Of interest, analysis of the CoMMpass dataset revealed significantly higher BCMA transcripts in patients with translocations involving MAP3K14 locus. Our findings highlight the impact of sBCMA levels and its chronicity of exposure on anti-BCMA TCE and CAR T activities in MM. We further identified structural genomic events driving TNFRSF17 over-expression and their correlation to sBCMA levels facilitating tumoral immunotherapeutic escape.

Comprehensive Characterisation of the Copy Number Landscape in Acute Lymphoblastic Leukemia Using Digitalmlpa

Introduction: Acute lymphoblastic leukemia (ALL) is a heterogeneous disease affecting various signaling pathways including cell differentiation and cell cycle control. ALL subtypes are defined by their genetic abnormalities, encompassing gross chromosomal aberrations, ploidy changes, translocations or rearrangements, regional or focal (intragenic) copy number alterations (CNAs), gene fusions and point mutations. Novel biomarkers for risk stratification are still being discovered, further mapping the diverse genetic ALL landscape. Testing for these newly identified genetic biomarkers and characterising the copy number (CN) landscape in ALL samples can provide useful new insights for risk stratification and patient-tailored treatment regimens. Methods: For multiplex detection of CNAs in ALL samples, a new test version of SALSA® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia was developed. Hereafter, this new test version will be referred to as D007 Acute Lymphoblastic Leukemia. This assay contains probes for CNA detection of 70 key genes and regions frequently altered in B-cell ALL and T-cell ALL ( Figure 1). In addition, this probemix contains digital karyotyping probes covering each chromosomal arm for the detection of gross chromosomal aberrations (except the p-arm of acrocentric chromosomes). A selection of these digital karyotyping probes also serve as reference probes. Finally, a set of single nucleotide polymorphism (SNP) probe pairs, targeting both alleles of each of the SNPs, was included for sample identification and guiding the correct CN baseline determination, facilitating optimal data normalisation in samples with complex karyotypes. To test the performance of this new test version of the probemix, DNA samples from 29 ALL cell lines were analysed. digitalMLPA-derived PCR amplicons were sequenced on a NextSeq 1000 Sequencing System (Illumina). FastQ files were analysed using Coffalyser digitalMLPA™ (MRC Holland) and a custom-built algorithm was used for copy number determination using SNP probe pairs. Results: digitalMLPA data of all 29 cell lines was analysed and compared to Cancer Cell Line Encyclopedia (CCLE) copy number data (Depmap, Copy Number Public 23Q2 download). CNAs were detected with D007 Acute Lymphoblastic Leukemia, including clinically relevant biomarkers such as the IKZF1 plus profile, PAX5alt, TCF3 fusions, focal 22q11.22 deletion (incl. VPREB1), intrachromosomal amplification of chromosome 21 (iAMP21), hypo- and hyperdiploidy. Other CNAs of emerging significance in ALL risk stratification included 6q15-q16 deletions, DNMT3B, CDKN1B and KRAS alterations. SNP probe pairs were effectively used to facilitate correct CN baseline determination, and results were confirmed using CCLE data (Depmap, CCLE_ABSOLUTE_combined 2019 download). Correct CN baseline detection is particularly important for samples with complex karyotypes, such as the P12-ICHIKAWA cell line. In this cell line, our data revealed that most chromosomal regions targeted by the SNP probe pairs had a copy number ≥3 ( Figure 2), consistent with the hypotetraploid karyotype previously described for this cell line. Conclusions: Using this new test version of SALSA® digitalMLPA™ D007 Acute Lymphoblastic Leukemia, numerous CNAs can be detected in a single reaction. Results obtained with this probemix showed a high correlation with CN data from the CCLE database in a panel of 29 ALL cell lines. digitalMLPA provides valuable insights into the CN of key target sequences relative to reference sequences in the genome. Moreover, our novel approach using pairs of probes that target both SNP alleles provides an additional layer of information, guiding CN and ploidy status determination in highly complex karyotypes. The SNP probe pair based approach for detection of CN levels in complex samples shows promising results and will be further validated in an independent cohort of 140 ALL patient samples. These results will be presented at the ASH meeting.

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma

The drivers of recurrence and resistance in ovarian high grade serous carcinoma remain unclear. We investigate the acquisition of resistance by collecting tumour biopsies from a cohort of 276 women with relapsed ovarian high grade serous carcinoma in the BriTROC-1 study. Panel sequencing shows close concordance between diagnosis and relapse, with only four discordant cases. There is also very strong concordance in copy number between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic copy number alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Copy number signatures are strongly correlated with immune cell infiltration, whilst diagnosis samples from patients with primary platinum resistance have increased rates of CCNE1 and KRAS amplification and copy number signature 1 exposure. Our data show that the ovarian high grade serous carcinoma genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers.

Cell-free DNA fragmentation profiling for therapeutic response monitoring in metastatic colorectal cancer.

e15664 Background: Currently available circulating cell-free DNA (cfDNA) assays require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the blood. Recently, we developed a tumor-agnostic, mutation-independent approach that utilizes low-coverage whole genome sequencing called DELFI (DNA evaluation of fragments for early interception) Tumor Fraction (DELFI-TF), a model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. Here, we report the results of DELFI-TF applied to a prospective cohort of patients with metastatic colorectal cancer (mCRC). Methods: Overall, 692 longitudinal plasma samples collected from 153 initially treatment-naive mCRC patients participating in the phase III CAIRO5 study (NCT02162563) were sequenced at low coverage and used for training and cross-validation. In patients with tumor-tissue-proven RAS/BRAF mutations, the tumor fractions were quantified as the cfDNA MAF of the RAS/BRAF variant measured by droplet digital PCR (ddPCR). Using fragment-sequencing statistics, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints to generate DELFI-TF scores. Changes in longitudinal DELFI-TF scores during first-line therapy (DELFI-TF slopes) were examined to predict treatment response and survival outcomes. Results: The DELFI-TF scores were strongly correlated with RAS/BRAF cfDNA MAF measured by ddPCR ( Pearson, r= 0.85, p< 0.001). Baseline DELFI-TF correlated with dimensions of liver metastases reported in CT scans ( Pearson, r= 0.49, p< 0.001) as well as clinical response, with pre-treatment levels significantly lower in patients with a later-confirmed partial or complete response ( Wilcoxon, p< 0.05). Patients with low or negative DELFI-TF slopes presented with longer progression-free survival in the overall study population (13.4 months vs 10.4 months, HR = 2.03, 95% CI 1.25 to 3.32, Log-rank p< 0.01) and in patients who experienced durable clinical benefit (16.7 months vs 13.3 months, HR = 2.24, 95% CI 1.1 to 4.55, Log-rank p= 0.023). Patients with low or negative DELFI-TF slopes also experienced significantly longer overall survival (59.4 months vs 29.1 months, HR = 3.05, 95% CI 1.58-5.90, Log-rank p< 0.001). Tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after liver metastases resection. Most patients detected as having a molecular relapse were diagnosed earlier than clinical recurrences identified by conventional imaging. Conclusions: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction.

Abstract CT109: First-in-class first-in-human phase 1 trial and translational study of the mono(ADP-ribose) polymerase-7 (PARP7) inhibitor RBN-2397 in patients with selected advanced solid tumors

Abstract Background: PARP7 is a stress-induced monoART that suppresses the cellular type I interferon (IFN) response following cytosolic nucleic acid sensing. RBN-2397 is a first-in-class PARP7 inhibitor that induces IFN and an adaptive immune response. The tumor-intrinsic immunomodulatory mechanism of RBN-2397 and preliminary antitumor activity in patients (pts) was demonstrated during dose escalation (Falchook, ASCO 2021; Kuplast-Barr, AACR 2022). Methods: Pts with solid tumors were treated with RBN-2397 at the RP2D of 200 mg BID in 3 expansion cohorts: squamous cell carcinoma of the lung (SCCL), head and neck squamous cell carcinoma (HNSCC), and hormone receptor-positive breast cancer (HR+ BC). Objectives of the expansion phase included safety, pharmacokinetics, pharmacodynamics, and antitumor activity. Results: As of 2 July 2022, 31 pts have been treated: SCCL (n=13), HNSCC (n=10), and HR+ BC (n=8). RBN-2397-related AEs (all grades >10%) included dysgeusia (42%, n=13), nausea (26%, n=8), fatigue (23%, n=7), with Grade 3 events of nausea and pleural infection (each n=1) and ALT/AST increase (n=2), and no Grade 4 events. No significant chronic toxicities were observed. The disease control rate in response-evaluable pts was 44% in SCCL (stable disease [SD] in 4/9 pts), 71% in HNSCC (RECIST partial response [PR] for 12+ months in 1/7; SD in 4/7), and 29% in HR+ BC (SD in 2/7). Biomarker analyses confirmed PARP7 mRNA expression in all baseline biopsies, with H-scores higher in tumor cells than in stromal cells (n=26, H-score range 66-256, P<0.0001). Four of 17 evaluable pts showed PARP7 focal copy number gains, and 2 had copy number gains that tracked with chromosome 3 copy number. One pt with HPV-negative HNSCC (nonsmoker; paranasal sinus primary) has an ongoing durable RECIST PR (−41%) for 12+ months. Tumor analysis of this responder confirmed PARP7 expression at baseline and SMARCB1 deletion, and demonstrated an excluded immune phenotype by IHC and MIBI-SCOPE. Analyses of paired tumor biopsies confirmed induction of adaptive immunity with ≥2-fold increases in CD8+ T cells and/or granzyme B expression in 10 (63%) of 16 pts across tumor types. Increases in immune checkpoint expression (PD-1 and LAG3 on T cells; PD-L1 on tumor cells) from 30% to 150% were observed in 3 (60%) of 5 pt tumor samples evaluated using MIBI-SCOPE, indicating the potential to prime tumors for immune checkpoint inhibitor therapy. These changes were independent of PARP7 copy number or PARP7 mRNA expression level at baseline. Conclusions: RBN-2397 was well tolerated at biologically active drug exposures, with preliminary antitumor activity observed. Paired tumor biopsy translational studies demonstrated the immunomodulatory mechanism of RBN-2397 and support the ongoing trial of RBN-2397 in combination with pembrolizumab (NCT05127590). Citation Format: Timothy A. Yap, Andres Cervantes, Gerald S. Falchook, Manish R. Patel, Dejan Juric, Saiama N. Waqar, Erin L. Schenk, Geoffrey Shapiro, Valentina Boni, Cesar A. Perez, Barbara Burtness, Yana G. Najjar, Fabricio Racca, Katerin Rojas, Kristy Kuplast-Barr, Kristen McEachern, Manoj Samant, Viviana Bozón, Sudha Parasuraman, Melissa Johnson. First-in-class first-in-human phase 1 trial and translational study of the mono(ADP-ribose) polymerase-7 (PARP7) inhibitor RBN-2397 in patients with selected advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT109.

Abstract 5714: Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer

Abstract BACKGROUND: Cell-free circulating tumor DNA (ctDNA) assays have been adopted to monitor therapeutic response in both early- and late-stage cancer. However, tests currently available require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the circulation. Recently, we developed a tumor-agnostic approach called DELFI Tumor Fraction (DELFI-TF), a Bayesian probabilistic model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. METHODS: Overall, 692 longitudinal plasma samples collected from 153 patients with initially unresectable colorectal cancer (CRC) liver-only metastases participating in the phase III study CAIRO5 (NCT02162563) were split across a training (RAS/BRAF mutant) and a validation cohort (RAS/BRAF wildtype). For all cell-free DNA (cfDNA) samples of patients within the training cohort (n=312), the tumor burden was quantified as the MAF of the tumor-tissue-proven RAS/BRAF variant measured by digital droplet PCR (ddPCR). Using cfDNA fragment-sequencing statistics from low-coverage WGS data, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints in the training cohort. Changes in DELFI-TF scores during first-line FOLFOX/FOLFIRI and bevacizumab therapy were assessed to predict disease response. A tissue-informed approach was applied to detect minimal residual disease in the subgroup of patients treated with curative intent (complete resection of the primary tumor and liver metastases). RESULTS: In the training cohort, DELFI-TF scores strongly correlated with RAS/BRAF MAF measured by ddPCR (r=0.85, p<0.001). DELFI-TF captured plasma tumor fractions associated with copy number changes when ddPCR failed to detect RAS/BRAF mutations. Before treatment initiation, DELFI-TF correlated with the sum of the largest diameters on CT scans according to RECIST1.1 (r=0.49, p<0.001). Patients with negative and positive changes in DELFI-TF more often exhibited response and progressive disease based on consecutive RECIST assessments, respectively. Median progression-free survival was 9.9 and 12.8 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.007). Overall survival was 28.8 and 58.9 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.001). In patients treated with curative intent, tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after metastasectomy. Most patients presenting molecular relapse were later diagnosed with clinical recurrence by conventional CT imaging. CONCLUSIONS: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with a performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction. Citation Format: Bahar Alipanahi, Iris van 't Erve, Keith Lumbard, Laurel K. Millberg, Zach Skidmore, Lorenzo Rinaldi, Jacob Carey, Jennifer Tom, Cornelis J. A. Punt, Nicholas C. Dracopoli, Gerrit A. Meijer, Robert B. Scharpf, Victor E. Velculescu, Remond Fijneman, Alessandro Leal. Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer. [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 5714.

Abstract 3861: c-MYC mediated resistance to trametinib plus hydroxychloroquine in pancreatic cancer is overcome by CDK4/6 and lysosomal inhibition

Abstract Pharmacological inhibition of KRAS>RAF>MEK>ERK signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDA). Interestingly, combined inhibition of MEK1/2 (with trametinib, T) plus autophagy (with chloroquine, CQ, or hydroxychloroquine, HCQ) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDA cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC-mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ resistant PDA tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of palbociclib plus hydroxychloroquine. Citation Format: Mark Silvis, Dilru Silva, Rohweder Riley, Sophia Schuman, Swapna Gudipaty, Amanda Truong, Jeffery Yap, Kajsa Affolter, Martin McMahon, Conan Kinsey. c-MYC mediated resistance to trametinib plus hydroxychloroquine in pancreatic cancer is overcome by CDK4/6 and lysosomal inhibition. [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 3861.

Abstract 1169: Genomic evolution of pancreatic cancer at single-cell resolution

Abstract As the breadth of genomic sequencing datasets increases, we can engage more directly with the evolutionary principles governing cancer progression. But as clonal evolution happens at the single-cell level, one must rely on strong assumptions when making conclusions with bulk-sequencing results, which fails in capturing clonal heterogeneity to its fullest. Herein we have developed and optimized a high-throughput, high-depth targeted single-nucleus DNA-seq (snDNA-seq) technique (doi.org/10.1101/2022.03.06.483206) for archival primary solid tumor samples. We focused on pancreatic ductal adenocarcinoma (PDAC), well known as one of the most lethal cancers, and sequenced over 200,000 single nuclei from 80 archival primary samples of 25 PDAC patients. The samples included both early- and late-stage diagnoses and multiregional sampling from primary tumors and metastasis to capture clonal heterogeneity on both temporal and spatial scales. With significant increase in sensitivity than bulk (down to mutations in 0.1% cells), we discovered thousands of novel mutations per sample on our 120,000 base-pair-long panel regions, suggesting a mutation rate higher than previously estimated. A small fraction of these mutations is in 1-10% single cells and are enriched in early-stage samples. They form mutually exclusive clones which functionally target key pathways including TGF-β, homologous recombination, suggesting subclonal evolution under positive selection at the early stage of cancer. Most novel mutations are in <1% single cells sampled and enabled us to measure convergent evolution and positive/negative selection within each tumor. We next revisited PDAC’s genomic evolution model established by bulk studies. It posits that PDAC often arises when KRAS hotspot mutation-bearing precursors acquire TP53 and/or CDKN2A inactivation through stepwise and punctuated evolution. While it assumes that TGF-β inactivating mutations are present in all cancer cells (clonal), we found that they are targeted in a highly subclonal manner; moreover, short mutations and focal copy number variations occur in a stepwise manner over time leading to the most “fit” genotype. In many PDACs whose bulk results show no alteration to the TGF-β pathway, snDNA-seq shows focal deletions that are likely below bulk’s sensitivity. Ongoing studies have begun to extend these analyses to longitudinal samples to study treatment response; normal pancreas tissues to study pancreas cells’ clonal evolution in aging and chronic disease conditions; blood samples to investigate circulating tumor cells in PDAC patients. Computational pipelines and analysis tools are being built as platform for more in-depth analyses. Overall, the high-throughput snDNA-seq technique brings genomic study of PDAC to a much higher resolution and holds the promise to not only inform precision medicine but also shed light on many fundamental questions on cancer evolution. Citation Format: Haochen Zhang, Palash Sashittal, Elias-Ramzey Karnoub, Benjamin J. Raphael, Christine A. Iacobuzio-Donahue. Genomic evolution of pancreatic cancer at single-cell resolution [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 1169.

Abstract P1-13-04: Potentiating Alpelisib in PI3K Pathway Overactive Triple Negative Breast Cancers

Abstract Potentiating Alpelisib in PI3K Pathway Overactive Triple Negative Breast Cancers David C. Boyd1,2, Amy L. Olex3, Tess Leftwich1, Nicole Hairr1, Alex K. Duong1, Narmeen S. Rashid1,3, Mohammad A Alzubi1,2, Holly Byers1, Aaron D. Valentine1, Julia E. Altman1, Emily Zboril1, Jacqueline M. Grible1, Madelyn Esquivel1, Scott A. Turner1, Andrea Ferreira-Gonzalez1, Mikhail G. Dozmorov5, J. Chuck Harrell1,2,6 1Department of Pathology, Virginia Commonwealth University; 2Integrative Life Sciences Program, VCU; 3Wright Center for Clinical and Translational Research, VCU; 4Department of Biology, University of Richmond; 5Department of Biostatistics, VCU; 6Massey Cancer Center, VCU. There is an urgent need for new therapeutic options for basal-like Triple Negative Breast Cancers (TNBC). To mirror the NCI-ComboMATCH study, and identify new synergistic drug combinations, we analyzed a set of 20 breast cancer patient-derived xenografts and 14 cell lines to identify targetable targets in each model. The Oncomine Comprehensive Assay v3 was performed to assess 161 genes for hotspot mutations, focal copy number variants, amplification/deletions, and RNA-fusion genes. Next, each PDX and their isogenic drug-resistant sublines were analyzed with bulk RNA-sequencing (217 samples) and cell single-cell RNA-sequencing (~100,000 cells). Of all NCI-MATCH defined targetable mutations, 37% of the models contained pathogenic PIK3CA amplifications or mutations. PIK3CA is one of the most common oncogenic aberrations identified in patients and the PI3K pathway is overactive in the majority of TNBCs, therefore we sought to target it across all models and identify the most efficacious synergistic drug partner. Short-term cultures of each PDX model were screened with a library of >1,000 FDA-approved/experimental drugs to identify compounds that were cytotoxic. Synergistic drug screens were then performed with each drug in combination with the PI3K inhibitor byl-719 (alpelisib) a current standard-of-care drug used for PI3K mutant ER+ disease. Synergism was identified using coefficient of drug interaction (CDI) calculations and 10 drugs were identified as synergistic across several models. Using several criteria, including clinical status and pathway analysis, 3 drugs were selected for CompuSyn-based synergism testing and each was confirmed to be synergistic in vitro. Testing with in vivo models of each drug combination has thus far confirmed that each is synergistic per CDI metrics. Those combinations are currently being tested for efficacy in the metastatic setting with a set of basal-like PDXs. Citation Format: David Boyd, Chuck Harrell. Potentiating Alpelisib in PI3K Pathway Overactive Triple Negative Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-04.

MYC-mediated resistance to trametinib and HCQ in PDAC is overcome by CDK4/6 and lysosomal inhibition.

Pharmacological inhibition of KRAS>RAF>MEK1/2>ERK1/2 signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDAC). Interestingly, combined inhibition of MEK1/2 (with trametinib [T]) plus autophagy (with chloroquine [CQ] or hydroxychloroquine [HCQ]) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDAC cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC-mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ-resistant PDAC tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of P/HCQ.