Deficient Cell Lines Research Articles

Photochemical Metal-Free synthesis and biological Assessment of isocryptolepine analogues targeting estrogen receptor Alpha in breast cancer cells

The aim of this study was to develop a new series of isocryptolepines and evaluate their antiproliferative and antiestrogenic activities on cancer cells. A series of isocryptolepine derivatives were synthesized using developed one-pot photochemical, metal-free protocol, employing readily available 2-arylindoles as starting compounds. The resulting isocryptolepines demonstrated (sub)micromolar inhibitory activity against selected breast cancer cell lines. The IC50 values ​​of lead compound 3c against hormone-dependent breast cancer types (MCF7 and T47D) were 0.3 μM and 0.12 μM, respectively, and significantly greater than 3 μM against estrogen receptor α (ERα)-deficient cell lines, MDA-MB-231 and HCC1954, respectively. To assess the antiestrogenic potency of compound 3c, MCF7 cells were transfected with a plasmid containing a luciferase reporter gene under the control of an estrogen-responsive element (ERE), creating the MCF7/ERE-LUC cell subline. In these cells, luciferase activity was induced by the natural ERα ligand, 17β-estradiol (E2). Compound 3c inhibited luciferase activity by 50 % at a concentration of 0.12 μM, highlighting its potent inhibitory effect on ERα. Molecular modeling further indicated that compound 3c could directly bind to ERα.Compound 3c induced apoptosis, as evidenced by PARP cleavage and downregulation of p-Bcl-2 and Bcl-2, and demonstrated synergistic effects in combination with the chemotherapeutic agent 5-fluorouracil. Compound 3c also showed selectivity towards hormone-dependent breast cancer cells, likely targeting ERα − a key driver in this cancer subtype. In summary, we report the development of a first-in-class antiestrogenic isocryptolepine with notable pro-apoptotic efficacy.

HADHA Regulates Respiratory Complex Assembly and Couples FAO and OXPHOS.

Oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) are key bioenergetics pathways. The machineries for both processes are localized in mitochondria. Secondary OXPHOS defects have been documented in patients with primary FAO deficiencies, and vice versa. However, the underlying mechanisms remain unclear. Intrigued by the observations that regulation of supercomplexes (SCs) assembly in a mouse OXPHOS deficient cell line and its derivatives is associated with the changes in lipid metabolism, a proteomics analysis is carried out and identified mitochondrial trifunctional protein (MTP) subunit alpha (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, HADHA) as a potential regulatory factor for SCs assembly. HADHA-Knockdown cells and mouse embryonic fibroblasts (MEFs) derived from HADHA-Knockout mice displayed both reduced SCs assembly and defective OXPHOS. Stimulation of OXPHOS induced in cell culture by replacing glucose with galactose and of lipid metabolism in mice with a high-fat diet (HFD) both exhibited increased HADHA expression. HADHA Heterozygous mice fed with HFD showed enhanced steatosis associated with a reduction of SCs assembly and OXPHOS function. The results indicate that HADHA participates in SCs assembly and couples FAO and OXPHOS.

The JAK inhibitor, Tofacitinib, Corrects the Overexpression of CEACAM6 and Limits Susceptibility to AIEC Caused by Reduced Activity of the IBD Associated Gene, PTPN2.

A cohort of patients with inflammatory bowel disease (IBD) exhibit expansion of the gut pathobiont, adherent-invasive E. coli (AIEC). Loss of activity of the IBD susceptibility gene, protein tyrosine phosphatase type 2 (PTPN2), results in dysbiosis of the gut microbiota both in human subjects and mice. Further, constitutive Ptpn2 knock-out (Ptpn2-KO) mice display expansion of AIEC compared to wildtype littermates. CEACAM6, a host cell surface glycoprotein, is exploited by AIEC to attach to and enter intestinal epithelial cells (IECs). Here, we investigate the role of IEC-specific PTPN2 in restricting AIEC invasion. Biopsies from IBD patients heterozygous (CT) or homozygous (CC) for the PTPN2 SNP (single nucleotide polymorphism) rs1893217 were processed for immunohistochemistry. HT-29 intestinal epithelial cells (IEC) were transfected with control shRNA (PTPN2-CTL), or a shRNA targeted towards PTPN2 (PTPN2-KD). The rs1893217 SNP was inserted (PTPN2-KI), or a complete knock-out of PTPN2 (PTPN2-KO) was generated, with CRISPR-Cas9 gene editing of Caco-2BBe IEC lines. Adherence and invasion assays were performed with either the human IBD AIEC isolate, LF82, or a novel fluorescent-tagged mouse adherent-invasive E. coli (mAIECred) at multiplicity of infection (MOI) of 10. IL-6 and the pan-JAK inhibitor tofacitinib were administered to interrogate JAK-STAT signaling. Protein expression was determined by western blotting and densitometry. CEACAM6 expression was elevated (colon and ileum) in IBD patients carrying the PTPN2 rs1893217 SNP (CT, CC) compared to wildtype (TT) IBD patients. HT-29 and Caco-2BBe cell lines deficient in PTPN2 expressed significantly higher levels of CEACAM6. Further, PTPN2-KI and PTPN2-KO cell lines also displayed greater adherence and invasion by AIEC LF82 and higher mAIECred invasion. CEACAM6 expression was further elevated after administration of IL-6 in PTPN2-deficient cell lines compared to untreated controls. Silencing of STAT1 and 3 partially reduced CEACAM6 protein expression. Tofacitinib significantly reduced the elevated CEACAM6 protein expression and the higher AIEC adherence and invasion in PTPN2-KI and PTPN2-KO cell lines compared to DMSO controls. Our findings highlight a crucial role for PTPN2 in restricting pathobiont entry into host cells. Our study also describes a role for the FDA-approved drug, tofacitinib (Xeljanz) in correcting the JAK-STAT-mediated over-expression of CEACAM6, used by pathobionts as an entry portal into host cells. These findings suggest a role for JAK-inhibitors in mitigating AIEC colonization in IBD-susceptible hosts.

Frequent CHD1 deletions in prostate cancers of African American men is associated with rapid disease progression

We analyzed genomic data from the prostate cancer of African- and European American men to identify differences contributing to racial disparity of outcome. We also performed FISH-based studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CHD1-deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. Subclonal deletion of CHD1 was nearly three times as frequent in prostate tumors of African American than in European American men and it associates with rapid disease progression. CHD1 deletion was not associated with HR deficiency associated mutational signatures or HR deficiency as detected by RAD51 foci formation. This was consistent with the moderate increase of olaparib and talazoparib sensitivity with several CHD1 deficient cell lines showing talazoparib sensitivity in the clinically relevant concentration range. CHD1 loss may contribute to worse disease outcome in African American men.

NMDtxDB: data-driven identification and annotation of human NMD target transcripts.

The nonsense-mediated RNA decay (NMD) pathway is a crucial mechanism of mRNA quality control. Current annotations of NMD substrate RNAs are rarely data-driven, but use generally established rules. We present a data set with four cell lines and combinations for SMG5, SMG6, and SMG7 knockdowns or SMG7 knockout. Based on this data set, we implemented a workflow that combines Nanopore and Illumina sequencing to assemble a transcriptome, which is enriched for NMD target transcripts. Moreover, we use coding sequence information (CDS) from Ensembl, Gencode consensus Ribo-seq ORFs, and OpenProt to enhance the CDS annotation of novel transcript isoforms. In summary, 302,889 transcripts were obtained from the transcriptome assembly process, out of which 24% are absent from Ensembl database annotations, 48,213 contain a premature stop codon, and 6433 are significantly upregulated in three or more comparisons of NMD active versus deficient cell lines. We present an in-depth view of these results through the NMDtxDB database, which is available at https://shiny.dieterichlab.org/app/NMDtxDB, and supports the study of NMD-sensitive transcripts. We open sourced our implementation of the respective web-application and analysis workflow at https://github.com/dieterich-lab/NMDtxDB and https://github.com/dieterich-lab/nmd-wf.

Generation of a ZEB2 deficient human iPSC line (KICRi002A-4)

The transcription factor ZEB2 is essential for early embryonic development. Using CRISPR/Cas9, we generated a ZEB2 deficient human iPSC cell line (KICRi002A-4), carrying a homozygous 790 bp deletion in ZEB2 that involves part of exon 5, intron 5 and part of exon 6. The deletion leads to markedly reduced levels of a truncated ZEB2 transcript. No ZEB2 protein was detected by immunopreciptation. The iPSC line expressed pluripotency markers and showed a capacity to differentiate into the three germ layers in vitro. Assessment of genomic integrity revealed a normal karyotype without detectable OFF-target editing. The iPSC line KICRi002A-4 thus offers a valuable resource to study the role of ZEB2 for the commitment and differentiation of various human cell lineages.

Increased Synthetic Cytotoxicity of Combinatorial Chemoradiation Therapy in Homologous Recombination Deficient Tumors

BackgroundHomologous recombination deficient (HRD) tumors are exquisitely sensitive to platinum-based chemotherapy and when combined with RT, leads to improved overall survival in multiple cancer types. Whether a subset of tumors with distinct molecular characteristics demonstrate increased benefit from cisplatin and RT (c-RT) is unclear. We hypothesized that HRD tumors, whether associated with BRCA mutations or genomic scars of HRD, exhibit exquisite sensitivity to c-RT, and that HRD may be a significant driver of c-RT benefit. MethodsSensitivity to c-RT was examined using isogenic and sporadic breast cancer cell lines. HRD was assessed using four assays: RT-induced Rad51 foci, a DR-GFP reporter assay, a genomic scar (large scale state transitions, LST), and clonogenic survival assays (CSA). Whole genome sequencing of 4 breast tumors from a phase 2 clinical trial of neoadjuvant c-RT in triple negative breast cancer (TNBC) was performed and defined HRD utilizing HRDetect. ResultsBRCA1/2 deficient cell lines displayed functional HRD based on the Rad51 functional assay, with c-RT to RT or cisplatin interaction ratios (IR) of 1.11 and 26.84 for the BRCA1 isogenic pair at 2uM Cisplatin and 6Gy, respectively. The highest LST lines demonstrated HRD and synthetic cytotoxicity to c-RT with IR at 2Gy and Cisplatin 20uM of 7.50, and the lowest LST line with IR of 0.65. Of 4 evaluable patients on the Phase 2 trial, one achieved pathologic complete response (pCR) with corresponding HRD based on multiple genomic scar scores including HRDetect and LST scores, compared with patients without pCR. ConclusionsHRD breast cancers, whether identified by BRCA1/2 mutation status, functional tests or mutational signatures, appear to be significantly more sensitive to c-RT compared to isogenic controls or tumors without HRD mutational signature. HRD tumors may be exquisitely sensitive to c-RT and warrants further clinical investigation to guide a precision oncology approach.

Identification of CSNK1D and KLK6 as two common upregulated genes present in BRCA1 mutated triple-negative breast cancer and ovarian epithelial carcinoma

Deficiency in the breast cancer type 1 (BRCA1) gene expression predisposes to triple-negative breast cancer (TNBC) and ovarian cancer (OC). We previously identified by Comparative Genomic Hybridization (CGH) array a gain in the 17q25.3 genomic region in 90% of the BRCA1 mutated TNBC tissues, where 17 genes were up-regulated. A second region (Chr19_45681759_54221324) was identified as the second most frequent gain in the BRCA1-mutated population and has not yet been described in the context of BRCA1 mutation. We thus aimed to validate the expression of the Casein kinase 1 delta (CSNK1D) gene of Chr17 in TNBC and OC cell lines and to investigate the expression of genes of Chr19 in TNBC cell lines and tissues as well as in OC cell lines. Expression level of the genes of the 17q25.3, 19q13.32,13.33 and 13.41 chromosomal regions was analyzed using RT-PCR in BRCA1 deficient TNBC and OC cell lines, as well as in 10 BRCA1-mutated TNBC tissues versus 10 wild type carriers. Our results revealed a significant upregulation of CSNK1D gene expression in BRCA1 deficient TNBC and OC cell lines when compared to control ones, and a significant aberration in the expression of the other six genes of Chr19 was observed. Interestingly, upregulation of kallikrein-related peptidase 6 (KLK6) was detected among the BRCA1 deficient TNBC (cell lines and tissues) and OC cell lines. In conclusion, our results suggested that CSNK1D and KLK6 expression levels could be very promising in the search for biomarkers for BRCA1 deficient TNBC and OC.

DNA Double-Strand Break Repair Kinetics after Exposure to Photons and Ions: A Systematic Review.

This study offers a review of published data on DNA double strand break (DSB) repair kinetics after exposure to ionizing radiation. By compiling a database, which currently includes 285 DNA DSB repair experiments utilizing both photons and ions, we investigate the impact of distinct experimental parameters on the kinetics of DNA DSB repair. Methodological differences and inconsistencies in reporting make the comparison of data generated by different research groups challenging. Nevertheless, by implementing filtering criteria, we can compare repair kinetics obtained with normal and tumor cells derived from human or animal tissues, as well as cells exposed to photons or ions ranging from hydrogen to iron ions. In addition, several repair curves of repair deficient cell lines were included. The study aims to provide researchers with a comprehensive overview of experimental factors that may confound results and emphasize the importance of precise reporting of experimental parameters. Moreover, we identify gaps in the literature that require attention in future studies, aiming to address clinically relevant questions related to radiotherapy. The database can be freely accessed at: https://github.com/weradstake/DRDNA.

Abstract 7148: Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR

Abstract Flap endonuclease 1 (FEN1) is a structure-specific metallonuclease that cleaves 5’ DNA flaps during replication and repair. FEN1 is an attractive target for development of anticancer therapeutics because it is overexpressed in many tumor types and has a large number of synthetic lethality partners including genes in Homologous Recombination (HR) pathway (Mengwasser et al., 2019; Guo et al., 2020). Utilizing fragment-based drug discovery approach we identified a novel metal-binding pharmacophore that binds to the two magnesium ions in the FEN1 active site. Further elaboration using fragment growth strategies resulted in highly potent and selective inhibitors. The current lead (BSM-1516) is ~65-fold more potent against FEN1 than its related enzyme Exonuclease 1 (Exo1) in biochemical assays (IC50 of 7 nM and 460 nM, respectively), an improvement of more than an order of magnitude in selectivity compared to earlier efforts. FEN1 target engagement in live cells was validated by cellular thermal shift assay (CETSA EC50 of 24 nM). Inhibition of FEN1 led to its increased association with chromatin in S-phase cells and recruitment of PARP1 enzyme. In clonogenic assay, BRCA2-deficient DLD1 cells were ~15-fold more sensitive to FEN1 inhibition than their isogenic BRCA2-wild-type counterparts (EC50 of 350 nM and 5 µM, respectively), confirming the increased susceptibility of HR deficient cancer cells to FEN1 inhibition. Treatment of BRCA2-deficient but not wild-type DLD1 cells with BSM-1516 resulted in cell cycle arrest and dose-dependent decrease of S-phase BrdU incorporation into DNA. Cell cycle arrest of BRCA2-deficient DLD1 cells was accompanied by DNA damage signaling and by accumulation of chromatin-bound RPA32, a marker of ssDNA. To explore synthetic lethal interactions of FEN1 with other DNA repair genes we performed FEN1-inhibitor-anchored CRISPR screen. This analysis revealed that in addition to HR pathway inactivation, genomic perturbations in EXO1, USP1 and PARP1 genes sensitized cells to FEN1 inhibition. Synergistic relationships of BSM-1516 and its combination potential were further explored in viability studies with a panel of DDR inhibitors (n=25) in BRCA2-proficient and deficient cell lines. Strong synergy was identified with multiple drug classes that included inhibitors of USP1 (KSQ-4279), PARP (Olaparib, Niraparib, Talazoparib, AZD5305), PARG (PDD 00017273) and ATR (AZD6738, VE-822, Elimusertib). In vitro ADME assays and in vivo PK studies showed that BSM-1516 has properties suitable for in vivo testing, either as a single agent or in combination with synergistic DDR inhibitors, an investigation that is currently underway. Citation Format: Jason Munguia, Sanjay Agarwalla, Elaine Guo, Dave Martin, Junhua Fan, Dave Lonergan, David Puerta, Zachary Zimmerman, Konstantin Taganov. Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR [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 7148.

Abstract 4538: Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma [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 4538.

Abstract 6597: CD40 agonism induces CD4 T cell mediated rejection of major histocompatibility complex class I deficient pancreatic tumors

Abstract Cytotoxic CD8 T cells are the canonical mediators of tumor clearance by the adaptive immune system. As such, current immunotherapies, including immune checkpoint blockade (ICB), target exhausted CD8 T cells with the hope of reinvigorating the CD8 T cells to drive tumor clearance. However, many tumors, including pancreatic ductal adenocarcinoma (PDAC), can evade CD8 T cell detection via downregulation of the antigen presentation molecule, major histocompatibility complex I (MHC I) as either primary or acquired resistance to immunotherapy. We have previously reported that agonistic CD40 antibody (αCD40), which promotes the maturation and licensing of dendritic cells to drive T cell priming, synergizes with ICB to induce tumor rejection dependent on both CD4 and CD8 T cells in mouse models of PDAC. Given the potential mechanisms of resistance to CD8 T cell immunity, we hypothesized that interrogation of the mechanisms underlying αCD40-induced CD4 T cell rejection of PDAC will reveal targets for improving CD4 T cell-directed immunotherapies. Here, we show that implantation of an MHC I deficient PDAC cell line derived from the genetically engineered KrasG12DTrp53R172HPdx1-Cre (KPC) mouse model results in normal tumor establishment and progression. Upon treatment with combined αCD40 and ICB, tumor growth was significantly decreased in a CD4, but not CD8, T cell-dependent manner. Using genetic knockout mice, we determined that tumor regression was perforin-independent and identified host expression of IFNγ as requisite for therapeutic efficacy. To identify the cellular subset mediating CD4 T cell-rejection of the tumor, we assessed the major histocompatibility complex II (MHC II) positive cells in the tumor microenvironment (TME), which possess the ability to directly synapse with CD4 T cells to drive anti-tumorigenic T cell programs. We determined that macrophage and myeloid cells were dispensable for treatment efficacy, however non-immune cells in the TME can also interact with CD4 T cells through expression of MHC II. Interrogation of stromal cells reveals an increase in the proportion of MHC II+ subsets after combined αCD40/ICB treatment, with a concomitant decrease in the proportion of MHC II+ tumor cells. These data support the notion that CD4 T cell control of tumors may be mediated by both direct and indirect mechanisms of tumor killing. Findings from this work will elucidate non-canonical mechanisms of CD4 T cell-mediated rejection of tumors and may be leveraged to discover novel translational targets for immunotherapy. Citation Format: Margaret E. Haerr, Samuel I. Kim, Charu Arora, Kyle P. Gribbin, Robert H. Vonderheide, Katelyn T. Byrne. CD40 agonism induces CD4 T cell mediated rejection of major histocompatibility complex class I deficient pancreatic tumors [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 6597.

Abstract 7237: Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy

Abstract BRCA1 and BRCA-2 proteins are involved in the repair of DNA damage such as double-strand breaks during S and G2 phases, by intervening in homologous recombination (HR) steps. Breast cancers arising from BRCA-1 mutation carriers (10% of BC) and from epigenetically silenced BRCA-1 (30% of sporadic BC) are associated with a lack of expression and/or function of BRCA-1, which induces genomic instability. Herein, we describe a new potent strategy combining mRNAs with a tumor selective peptide nanocarrier, to selectively rescue BRCA-1 function as potential therapeutic approach in cancers. Proprietary BRCA-1 derived mRNA were complexed with proprietary short amphipathic peptides and the resulting nanoparticles were evaluated on several breast cancer cells, harboring different BRCA-1 alterations. Sensitivity to Veliparib (PARPi) following the nanoparticle treatments were evaluated on PARPI resistant and PARPi sensitive cells. The recruitment of RAD51 to sites of double stranded DNA break was quantified by immunofluorescence. In-vivo efficacy of varying sequences of BRCA-1 mRNA-peptide nanoparticles (2.0 mg/kg, weekly, IV; ADGN-901, -902, -903) and veliparib (50mg/kg, daily, oral) were evaluated on breast cancer (HCC1937-BRCA-1 deficient, PARPi resistant) mouse xenografts. We showed that ADGN-901 inhibits the proliferation of BRCA-1 deficient cell lines from 20 to 60% depending on the type of BRCA-1 alterations. ADGN-902, specifically reduces the proliferation of cells expressing N-terminal truncated BRCA-1, by 50 to 60% and ADGN-903 inhibits proliferation >60% of all cells harboring defects in BRCA-1. Interestingly, ADGN-901 and ADGN-903 were able to resensitize the PARPi resistant cells to veliparib inhibition. We demonstrated that ADGN-903 restores BRCA-1 function in homologous recombination. ADGN-903 prevents RAD51 recruitment to the damage site by reducing RAD51 protein level as well as formation of RAD51 foci following irradiation. IV administration of ADGN-901 and ADGN-903 (2.0 mg/kg) had a strong inhibitory effect on HCC-1937 tumor growth while ADGN-902 and veliparib were inactive. ADGN-901 reduced tumor growth by 95% and ADGN-903 abolished tumor growth with strong tumor regressions. ADGN-901 and ADGN-903, were also evaluated on large tumors of about 500-600 mm3 and were highly effective in preventing further progression and achieving shrinkage of large tumors. ADGN-901 and ADGN-903 treatments are well tolerated, without inducing clinical toxicity. BRCA-1 mRNA-peptide nanoparticles are effective in rescuing BRCA-1 function and inducing tumor regressions in BRCA-1 altered breast cancer cells and tumors. Our study provides a proof-of-concept that restoration of wild type BRCA-1 expression could be a potent therapeutic strategy for BRCA-1 altered tumors whether or not they are PARPi resistant. Citation Format: Gilles Divita, Audrey Grunenberger, Léa Cabrera, Nathalie Durany, Neil Desai. Peptide-mRNA complex mediated restoration of BRCA-1 tumor suppressor function in BRCA-1 mutated cancers as a new therapeutic strategy [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 7237.

Abstract 7061: Targeting metabolic vulnerabilities in ARID1A/B dual-deficient dedifferentiated endometrial carcinoma

Abstract Background: Dedifferentiated endometrial carcinoma (DDEC) is an aggressive endometrial carcinoma defined histologically by undifferentiated carcinoma juxtaposed against stage 1 or 2 endometrial adenocarcinoma. DDEC responds poorly to conventional platinum/taxane-based chemotherapy, particularly in the case of extrauterine spread. This highlights a need to develop novel therapies for individuals with DDEC. Genetically, a third of DDEC cases have co-inactivating mutations of ARID1A and ARID1B. These genes encode core subunits of the sucrose/switch non-fermentable (SWI/SNF) complex that regulate transcription. Furthermore, dual loss of ARID1A/B is believed to play a role in driving DDEC tumor development. We hypothesize that ARID1A/B dual-deficiency in DDEC generates unique genetic deficiencies that allow for the development of novel therapies for this cancer. We approached this hypothesis by first conducting a preliminary analysis of the Cancer Dependency Map (DepMap), which has identified vulnerabilities in mitochondrial functioning in ARID1A/B dual-deficient DDECs. As such, we aimed to understand the role of mitochondrial oxidative phosphorylation (OXPHOS) and redox homeostasis in ARID1A/B dual-deficient DDEC to create new treatment options. Methods: A panel of ARID1A/B dual-deficient and proficient endometrial cancer cell lines were treated with OXPHOS inhibitor IACS-010759 and reactive oxidative species (ROS)-inducing agent elesclomol to identify shifts in drug response between the two groups. To account for genomic differences between the cell lines, we also treated an ARID1B knock-out isogeneic cell line with the drugs. Additionally, changes in metabolism in the endometrial cancer cell lines were measured using Seahorse metabolic flux assays to determine if ARID1A/B dual-deficient cell lines have an increased dependency on OXPHOS for cell growth. Results: While the Seahorse assays revealed no significant difference in OXPHOS and glycolysis activities between the two groups, drug sensitivity assays demonstrated that ARID1A/B dual-deficient cancers are more sensitive to OXPHOS inhibition and increased ROS production than the proficient cell lines. Depletion of ARID1B in an ARID1A deficient cell line sensitizes the cells to both drugs. Furthermore, IACS-010759 significantly suppressed the growth of ARID1A/B-deficient DDEC xenograft tumor growth. Conclusion: ARID1A/B-dual deficient cancer cells rely on mitochondria function for survival. The underlying mechanisms are currently under investigation. Citation Format: Rebecca Ho, Eunice Li, Chae Young Shin, Shary Chen, David Huntsman, Yemin Wang. Targeting metabolic vulnerabilities in ARID1A/B dual-deficient dedifferentiated endometrial carcinoma [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 7061.

Abstract P23: RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma

Abstract P23: RHOA inactivation subverts IFN-γ response and promotes antigen escape in CAR-T resistant diffuse large B-cell lymphoma

Abstract PR018: Adaptive local fitness landscapes for aneuploid karyotypes

Abstract Aneuploidy, which occurs in most solid tumors, dramatically alters the expression of many genes which in turn has broad phenotypic effects. Recent evidence suggests that specific aneuploid karyotypes confer a selective advantage. However, various questions remain unanswered regarding the magnitude of these selective advantages, as well as the repeatability and predictability of karyotype evolution. We developed ALFA-K (Adaptive Local Fitness landscapes for Aneuploid Karotypes) – the first approach to infer karyotype fitness landscapes in a context-dependent manner. Based on the dynamics of just a few subclones derived from longitudinal single cell sequencing data, our method can extrapolate the fitness of thousands of karyotypes. We validate the predictive power of our method on data from several P53 deficient cell lines which exhibited substantial subclonal evolution across multiple passages in vivo or in vitro. Based on topological analyses of the inferred fitness landscapes we investigated how mis-segregation rate itself shapes the stringency of karyotype evolution and whether karyotype evolution is subject to diminishing returns epistasis, sign epistasis or reciprocal sign epistasis. We adapted metrics from geo-statistics to study the fitness landscape of a P53 deficient 184-hTERT diploid breast epithelial cell line that underwent whole genome doubling (WGD) and found support for the hypothesis that WGD benefits tumor cells by enhancing their robustness against chromosomal instability. The stark phenotypic consequences of karyotype changes emphasize the value of predicting the evolutionary trajectory of karyotype composition when exposing cells to a new environment. Our work contributes to accumulating evidence that karyotype evolution is predictable. Citation Format: Richard J. Beck, Noemi Andor. Adaptive local fitness landscapes for aneuploid karyotypes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR018.

Abstract PR005: Identification of DNA Damage Repair genes controlling the immune landscape of breast tumors via spatial functional genomics

Abstract Immune checkpoint blockade (ICB) has revolutionized the field of cancer immunology, yet only 17-20% of patients who receive this treatment derive any clinical benefit. There is an unmet need to either identify the subset of patients who would respond to ICB or identify targets for combination therapies. By assessing frequently mutated pathways in these patients, we can identify potential biomarkers and candidates for combinatorial therapy. One such pathway is the DNA Damage Response (DDR) pathway. Faults in DDR pathways can lead to genome instability, higher tumor mutational burden (TMB), and accumulation of neoantigens in tumors. Consequently, tumors with one or more deficiencies in DDR (DDRd) have been shown to respond better to therapy and are associated with increased immune cell infiltration. However, previous work from our lab has shown that different gene alterations, even in the same DDR pathway, can polarize immune outcomes differently. To address this issue, we employed Perturb-map, a first-of-its-kind functional genomics approach, which utilizes protein barcodes (pro-Codes) to enable spatial resolution of CRISPR screens. We generated a Pro-Code/CRISPR library targeting frequently mutated DDR genes and introduced the library into 4T1 breast cancer cells, a syngeneic model of triple negative breast cancer. We introduced the cells into immunocompetent mice and performed multiplex imaging on metastatic lesions in the lung to spatially resolve each gene knockout tumor. We observed that loss of Rbbp8 and Polq led to larger tumors, whereas loss of Pms2 led to smaller tumors. Strikingly, Rbbp8 knockout tumors were also less infiltrated by CD4 T cells compared to control tumors. This system was also used to find therapeutic targets by performing synthetic lethality screens using mass cytometry. We transduced the library in isogenic Brca2 deficient 4T1 cells along with their wildtype (WT) counterparts and looked for enrichment or loss of pro-Codes across the different cell lines. These screens consistently identified known synthetic lethality partners like Parp1 in the Brca2 deficient cell lines and identified additional targets like Mre-11a, Bard1 and Rbbp8, which are being profiled further. Taken together, we have developed a scalable system to study the effect of DDR mutations on tumor cell intrinsic and extrinsic properties, which can not only help characterize immune outcomes but also identify potential therapeutic partners. This system proves to be a useful tool that can be leveraged to profile a number of different genes across multiple cancer types. Citation Format: Prerna Suri, Juhana Habib, Gurkan Mollaoglu, Luisanna Pia, Alessia Baccarini, Brian Brown, Robert Samstein. Identification of DNA Damage Repair genes controlling the immune landscape of breast tumors via spatial functional genomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR005.

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Caspase-mediated AURKA cleavage at Asp132 is essential for paclitaxel to elicit cell apoptosis.

Background: Aurora kinase A (AURKA) is a potent oncogene that is often aberrantly expressed during tumorigenesis, and is associated with chemo-resistance in various malignancies. However, the role of AURKA in chemo-resistance remains largely elusive. Methods: The cleavage of AURKA upon viral infection or apoptosis stimuli was assesed by immunoblotting assays in several cancer cells or caspase deficient cell line models. The effect of AURKA cleavage at Asp132 on mitosis was explored by live cell imaging and immunofluorescence staining experiments. The role of Asp132-cleavage of AURKA induced by the chemotherapy drug paclitaxel was investigated using TUNEL, immunohistochemistry assay in mouse tumor xenograft model and patient tissues. Results: The proteolytic cleavage of AURKA at Asp132 commonly occurs in several cancer cell types, regardless of viral infection or apoptosis stimuli. Mechanistically, caspase 3/7/8 cleave AURKA at Asp132, and the Asp132-cleaved forms of AURKA promote cell apoptosis by disrupting centrosome formation and bipolar spindle assembly in metaphase during mitosis. The AURKAD132A mutation blocks the expression of cleaved caspase 3 and EGR1, which leads to reduced therapeutic effects of paclitaxel on colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model and cancer patients. Conclusions: This study reveals that caspase-mediated AURKAD132 proteolysis is essential for paclitaxel to elicit cell apoptosis and indicates that AURKAD132 is a potential key target for chemotherapy.

Abstract A115: mTOR targeting in STK11 deficient Non-Small Cell Lung Cancer (NSCLC): final results, pre-clinical rationale and biomarker analysis of a phase II trial of the mTORC1/2 inhibitor vistusertib in STK11 deficient lung adenocarcinoma (NLMT B2)

Abstract Background: STK11 loss of function is common in lung adenocarcinoma (LUAD). STK11 deficient LUAD demonstrates mTOR activation, increased HIF-1a expression and GLUT1 up-regulation, resulting in increased glucose metabolism. STK11 loss often co-occurs with KRAS mutation. KRAS and STK11 co-mutations synergise to drive metabolic re-programming and heightened glucose dependency, reversible with mTORC1/mTORC2 inhibition. mTORC2 is a central node driving enhanced glycolysis independent of Raptor and HIF-1α. mTORC1/2 inhibition suppresses pS6K, p4EBP1, HIF1a and GLUT1 in STK11/KRAS double mutant NSCLC cells and suppresses growth in vivo. Vistusertib (AZD2014) is a selective mTORC1/2 inhibitor. We report the results of outcomes of LUAD patients with STK11 loss+/-KRAS mutation treated with vistusertib, the first study to investigate the impact of mTOR inhibition in STK11 deficient LUAD. Results: 19 patients (17 per-protocol) had STK11 loss without concomitant KRAS mutation (cohort B2S). 30 patients had STK11 loss with concomitant KRAS mutation (26 per-protocol) (cohort B2D). The confirmed objective response rate (95% credible interval) was 3.8% (0.1 – 18.5) for B2S, 9.8% (2.4 – 24.3) for B2D. Durable clinical benefit ((DCB), defined as no progression at 4th 6 weekly on treatment CT assessment) rate was 14.6% (3.6 – 34.7) for B2S, 24.4% (11.1 – 42.3) for B2D. Tumour growth inhibition (median % change from baseline (range)) in those with DCB was -18.5% (-48.0, 3.0) and in those with progression <3 months/death +10.5% (-6.0, 42.0). Median PFS was 2.29 months (1.47 – 3.85) for B2S, 2.77 months (1.91 – 4.22) for B2D. Median OS was 9.84 months (6.23 – 16.85) for B2S, 6.08 months (4.20 – 9.26) for B2D. Clinically achievable concentrations of vistusertib achieve dose-dependent inhibition of STK11 deficient LUAD cell line viability in vitro. In contrast, IC50 for everolimus (mTORC1-only inhibition) exceeds pharmacologically achievable doses in all tested STK11 deficient cell lines. Rebound FOXO and Akt phosphorylation is observed after 48 hours of vistusertib treatment, with parallel activation of ERK. Longitudinal ctDNA from B2S/B2D were analysed using the Illumina TSO500 ctDNA platform. Using conventional p<0.05 for significance, 2 genes were enriched at progression versus baseline: SMARCA4 (3/36 pre, 11/27 post, p=0.005) and FOXP1 (0/36 pre, 4/27 post, p=0.029). TRAF2 mutations were non-significantly enriched post-treatment (1/36 pre, 5/27 post, P = 0.076). Conclusions: Despite strong rationale for mTORC1/2 inhibition in STK11 deficient LUAD, activity of vistusertib is modest. Pre-clinical work suggests that STK11 mutant LUAD cell lines are not exempt from relief of feedback inhibition during prolonged mTORC1/2 inhibition, driving Akt and FOXO phosphorylation. Further, longitudinal ctDNA analysis suggests that vistusertib treatment may drive selection for SMARCA4 mutations, which appears to be a common mechanism of resistance to targeted therapies. HLR and PF contributed equally. LB and GM contributed equally. Citation Format: Helen L Robbins, Peter Fletcher, Joshua Savage, Manita Mehmi, Yvonne Summers, Alastair Greystoke, Noelle O’Rourke, Sanjay Popat, Pooja Jain, James Spicer, Judith Cave, Paul Shaw, David Gilligan, Danielle Power, Dean Fennel, Andrew D Beggs, Lucinda Billingham, Gary Middleton. mTOR targeting in STK11 deficient Non-Small Cell Lung Cancer (NSCLC): final results, pre-clinical rationale and biomarker analysis of a phase II trial of the mTORC1/2 inhibitor vistusertib in STK11 deficient lung adenocarcinoma (NLMT B2) [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 A115.