Vulnerability Of Cancer Cells Research Articles

Irradiation accelerates SARS-CoV-2 infection by enhancing sphingolipid metabolism.

Cancer patients who receive radiotherapy have a high risk of severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2)infection, but the concrete reason remains unclear. Herein, we investigated the influence of irradiation on the vulnerability of cancer cells to SARS-CoV-2 using S pseudovirions and probed the underlying mechanism via RNA-seq and other molecular biology techniques. Owing to the enhancement of sphingolipid metabolism, irradiation accelerated pseudovirion infection. Mechanistically, irradiation induced the expression of acid sphingomyelinase (ASM), which catalyses the hydrolysis of sphingomyelin to ceramide, contributing to lipid raft formation and promoting SARS-CoV-2 invasion. Inhibition of lipid raft formation with methyl-β-cyclodextrin (MβCD) or the tyrosine kinase inhibitor genistein and ASM suppression through small interfering RNA or amitriptyline (AMT) treatment abolished the enhancing effect of irradiation on viral infection. Animal experiments supported the finding that irradiation promoted SARS-CoV-2 S pseudovirion infection in A549 cell tumour-bearing BALB/c nude mice, whereas AMT treatment dramatically decreased viral infection. This study discloses the role of sphingolipid metabolism in irradiation-induced SARS-CoV-2 infection, thus providing a potential target for clinical intervention to protect patients receiving radiotherapy from COVID-19.

Spatially resolved phosphoproteomics reveals fibroblast growth factor receptor recycling-driven regulation of autophagy and survival

Receptor Tyrosine Kinase (RTK) endocytosis-dependent signalling drives cell proliferation and motility during development and adult homeostasis, but is dysregulated in diseases, including cancer. The recruitment of RTK signalling partners during endocytosis, specifically during recycling to the plasma membrane, is still unknown. Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b) recycling, we reveal FGFR signalling partners proximal to recycling endosomes by developing a Spatially Resolved Phosphoproteomics (SRP) approach based on APEX2-driven biotinylation followed by phosphorylated peptides enrichment. Combining this with traditional phosphoproteomics, bioinformatics, and targeted assays, we uncover that FGFR2b stimulated by its recycling ligand FGF10 activates mTOR-dependent signalling and ULK1 at the recycling endosomes, leading to autophagy suppression and cell survival. This adds to the growing importance of RTK recycling in orchestrating cell fate and suggests a therapeutically targetable vulnerability in ligand-responsive cancer cells. Integrating SRP with other systems biology approaches provides a powerful tool to spatially resolve cellular signalling.

Predictive markers for efficiency of the amino-acid deprivation therapies in cancer

Amino acid deprivation therapy (AADT) is a promising strategy for developing novel anticancer treatments, based on variations in metabolism of healthy and malignant cells. L-asparaginase was the first amino acid-degrading enzyme that received FDA approval for the treatment of acute lymphoblastic leukemia (ALL). Arginase and arginine deiminase were effective in clinical trials for the treatment of metastatic melanomas and hepatocellular carcinomas. Essential dependence of certain cancer cells on methionine explains the anticancer efficacy of methionine-g-lyase. Along with significant progress in identification of metabolic vulnerabilities of cancer cells, new amino acid-cleaving enzymes appear as promising agents for cancer treatment: lysine oxidase, tyrosine phenol-lyase, cysteinase, and phenylalanine ammonia-lyase. However, sensitivity of specific cancer cell types to these enzymes differs. Hence, search for prognostic and predictive markers for AADT and introduction of the markers into clinical practice are of great importance for translational medicine. As specific metabolic pathways in cancer cells are determined by the enzyme expression, some of these enzymes may define the sensitivity to AADT. This review considers the known predictors for efficiency of AADT, emphasizing the importance of knowledge on cancer-specific amino acid significance for such predictions.

MRNA cytidine N4‐acetylation by NAT10 modulates lipid utilization in breast cancer

mRNA cytidine N4‐acetylation by NAT10 modulates lipid utilization in breast cancer

Targeting replication stress in cancer therapy.

Replication stress is a major cause of genomic instability and a crucial vulnerability of cancer cells. This vulnerability can be therapeutically targeted by inhibiting kinases that coordinate the DNA damage response with cell cycle control, including ATR, CHK1, WEE1 and MYT1 checkpoint kinases. In addition, inhibiting the DNA damage response releases DNA fragments into the cytoplasm, eliciting an innate immune response. Therefore, several ATR, CHK1, WEE1 and MYT1 inhibitors are undergoing clinical evaluation as monotherapies or in combination with chemotherapy, poly[ADP-ribose]polymerase (PARP) inhibitors, or immune checkpoint inhibitors to capitalize on high replication stress, overcome therapeutic resistance and promote effective antitumour immunity. Here, we review current and emerging approaches for targeting replication stress in cancer, from preclinical and biomarker development to clinical trial evaluation.

Selective vulnerability of ARID1A deficient colon cancer cells to combined radiation and ATR-inhibitor therapy.

ARID1A is frequently mutated in colorectal cancer (CRC) cells. Loss of ARID1A function compromises DNA damage repair and increases the reliance of tumor cells on ATR-dependent DNA repair pathways. Here, we investigated the effect of ionizing radiation (IR), in combination with ATR inhibitors (ATRi) in CRC cell lines with proficient and deficient ARID1A. The concept of selective vulnerability of ARID1A deficient CRC cells to ATRi was further tested in an ex vivo system by using the ATP-tumor chemosensitivity assay (ATP-TCA) in cells from untreated CRC patients, with and without ARID1A expression. We found selective sensitization upon ATRi treatment as well as after combined treatment with IR (P<0.001), especially in ARID1A deficient CRC cells (P <0.01). Knock-down of ARID1B further increased the selective radiosensitivity effect of ATRi in ARID1A negative cells (P<0.01). Mechanistically, ATRi abrogates the G2 checkpoint (P<0.01) and homologous recombination repair (P<0.01) in ARID1A deficient cells. Most importantly, ex-vivo experiments showed that ATRi had the highest radiosensitizing effect in ARID1A negative cells from CRC patients. Collectively, our results generate pre-clinical and clinical mechanistic rationale for assessing ARID1A defects as a biomarker for ATR inhibitor response as a single agent, or in a synthetic lethal approach in combination with IR.

HMGA2 drives the IGFBP1/AKT pathway to counteract the increase in P27KIP1 protein levels in mtDNA/RNA-less cancer cells.

Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; therefore, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.

Distinct mechanism of cervical cancer cell death caused by the investigational new drug SHetA2.

Drug-targetable vulnerabilities of cancer cells include their dependence on heat shock proteins (HSPs) to support elevated mitochondrial metabolism and counteract cell death factors. The investigational new drug SHetA2 targets these vulnerabilities in ovarian and endometrial cancer cells by disrupting complexes of the mortalin HSP with its client proteins (mitochondrial support proteins, metabolic enzymes, p53) leading to mitochondrial leakage of cytochrome c and apoptosis-inducing factor (AIF), and caspase-dependent apoptosis. Our objective was to evaluate the roles of mitochondrial damage and another SHetA2-target HSP protein, cytoplasmic heat shock cognate 70 (hsc70), in the mechanism of SHetA2 killing of cervical cancer cells. Cervical cancer cells responded to SHetA2 with excessive mitophagy that did not deter AIF leakage into the cytoplasm. Then, hsc70 was unable to prevent cytoplasmic AIF nuclear translocation and promotion of DNA damage and cell death, because SHetA2 disrupted hsc70/AIF complexes. The Cancer Genome Atlas analysis found that overexpression of hsc70, but not mortalin, was associated with worse cervical cancer patient survival. Use of specific inhibitors documented that AIF and mitophagy, but not caspases, contributed to the mechanism of SHetA2-induced cell death in cervical cancer cells. As validation, excessive mitophagy and lack of caspase activation were observed in SHetA2-inhibited xenograft tumors.

In This Issue

In This Issue

The molecular network of the proteasome machinery inhibition response is orchestrated by HSP70, revealing vulnerabilities in cancer cells.

Proteasome machinery is a major proteostasis control system in human cells, actively compensated upon its inhibition. To understand this compensation, we compared global protein landscapes upon the proteasome inhibition with carfilzomib, in normal fibroblasts, cells of multiple myeloma, and cancers of lung, colon, and pancreas. Molecular chaperones, autophagy, and endocytosis-related proteins are the most prominent vulnerabilities in combination with carfilzomib, while targeting of the HSP70 family chaperones HSPA1A/B most specifically sensitizes cancer cells to the proteasome inhibition. This suggests a central role of HSP70 in the suppression of the proteasome downregulation, allowing to identify pathways impinging on HSP70 upon the proteasome inhibition. HSPA1A/B indeed controls proteasome-inhibition-induced autophagy, unfolded protein response, and endocytic flux, and directly chaperones the proteasome machinery. However, it does not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, targeting of NRF1 proves effective in decreasing the viability of cancer cells with the inhibited proteasome and HSP70.

A high-throughput drug screen reveals means to differentiate triple-negative breast cancer

Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC.

Pan-cancer proteomic map of 949 human cell lines.

SummaryThe proteome provides unique insights into disease biology beyond the genome and transcriptome. A lack of large proteomic datasets has restricted the identification of new cancer biomarkers. Here, proteomes of 949 cancer cell lines across 28 tissue types are analyzed by mass spectrometry. Deploying a workflow to quantify 8,498 proteins, these data capture evidence of cell-type and post-transcriptional modifications. Integrating multi-omics, drug response, and CRISPR-Cas9 gene essentiality screens with a deep learning-based pipeline reveals thousands of protein biomarkers of cancer vulnerabilities that are not significant at the transcript level. The power of the proteome to predict drug response is very similar to that of the transcriptome. Further, random downsampling to only 1,500 proteins has limited impact on predictive power, consistent with protein networks being highly connected and co-regulated. This pan-cancer proteomic map (ProCan-DepMapSanger) is a comprehensive resource available at https://cellmodelpassports.sanger.ac.uk.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer

BackgroundTargeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L.MethodsWe combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes.ResultsDot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells.ConclusionsIdentification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis.

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.

Abstract 3624: Non-canonical proteins are cancer cell vulnerabilities in diverse malignancies

Abstract In the 20 years since the completion of the Human Genome Project, cancer biology remains rooted in the assumption that the human genome encodes ~20,000 protein-coding genes. Yet, I and others have shown that thousands of “non-canonical” open reading frames (ncORFs) populate the human genome, potentially representing a dramatic expansion of the cancer proteome. Despite their abundance, little is known about the role of ncORFs as cancer driver genes. We developed functional genomics approaches to pursue this question across human cancers. To determine whether ncORFs represent biologically active proteins, we experimentally interrogated 553 candidates selected from ncORF datasets. Of these, 257 (46%) showed evidence of stable protein expression using multiple assays, and 401 (72%) induced gene expression changes when expressed in cancer cell lines. The bioactivity of ncORFs was dependent on their ability to translate a protein: mutation of the ORF start codon prevented induction of gene expression changes observed with the wild type ncORF in 48 of 51 (94%) cases. Using custom CRISPR/Cas9 knock-out screens targeting &amp;gt;2,000 ncORFs in 20 cancer cell lines, we found that genomic knock-out of approximately 10% of ncORFs induced viability defects in cancer cells. We focused on two candidates for functional studies. In breast cancer, we described G029442 - renamed glycine-rich extracellular protein-1 (GREP1) - as a secreted protein that is highly expressed and prognostic for poor patient outcomes. Knock-out of GREP1 in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. In medulloblastoma, we found that MYC-driven medulloblastoma cells are enriched for bioactive upstream ORFs (uORFs) that are encoded within the 5’ untranslated regions of mRNAs. We validated the ASNSD1 uORF as a top genetic vulnerability in multiple models of medulloblastoma, and its overexpression is sufficient to increase neural stem cell proliferation. Mechanistically, ASNSD1 uORF promotes a MYC-associated cellular program and interacts with the multiprotein prefoldin complex, which is required for tumors to maintain post-transcriptional regulation. Our work supports a generalizable principle that ncORFs commonly encode biologically-active proteins in diverse malignancies. Ongoing investigation of ncORFs therefore represents a new frontier in cancer research with the potential to define the next generation of therapeutic target genes. Citation Format: John R. Prensner, Ian Yannuzzi, Karl Clauser, Karsten Krug, Oana Enache, Adam Brown, Amy Goodale, David E. Root, Pratiti Bandopadhayay, Todd Golub. Non-canonical proteins are cancer cell vulnerabilities in diverse malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3624.

Abstract LB544: Targeting adaptive metabolic program as a novel treatment approach for TKIs-failed ALK-positive NSCLCs

Abstract Introduction: Acquired resistance to ALK-tyrosine kinase inhibitors (ALK-TKIs) treatment, particularly target-off resistance, remains a clinical challenge for ALK-rearranged non-small cell lung cancer (NSCLC). To explore novel vulnerabilities of ALK TKI-resistant cancer cells, we focused on their distinct metabolic pathways for growth and survival. Experimental Design: To investigate metabolic pathways in resistance mechanisms, we generated ALK-TKIs -acquired-resistant in vitro/vivo models. We screened metabolite mechanisms using metabolite assay kit, Seahorse Extracellular Flux Analyzer, real-time PCR, western blot, RNA-seq in resistant models. Results: Through an integrated transcriptomic and metabolic assay screening approach, we identified the enhanced reliance on glutamine metabolism in target-off ALK-TKIs-resistant cells. Specifically, resistant cells were characterized by upregulation of glutaminase 1 (GLS1), a mitochondrial enzyme hydrolyzing glutamine into glutamate, simultaneously with downregulation of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this metabolic state intensively accelerates glutaminolysis and subsequent mitochondrial glutamine-derived aspartate synthesis, resulting in TKI resistance by reinforcing antioxidant capacity with increase of NADPH and glutathione. Mechanistically, GLS1 inhibition elicited a marked reduction of cell growth with increase of reactive oxygen species (ROS) in resistant cells, which was restored by supplementation of exogenous aspartate. The antitumor activity of GLS1 inhibition against resistant tumor cells was further validated in in vivo experiments, patient-derived xenograft (PDX) and EML4-ALK transgenic mice. More importantly, glutaminase inhibitor CB-839 enhanced the therapeutic efficacy of anti-PD-L1 treatment in immune checkpoint blockade (ICB)-resistant EML4-ALK transgenic mice. Conclusion: Our findings highlight a new metabolic vulnerability of ALK-TKIs resistant tumors and provide a rationale for targeting GLS1 as a potential treatment option to overcome ALK-TKIs resistance. Citation Format: You Won Lee, Hun Mi Choi, Seung Yeon Oh, Eun Ji Lee, Kyoung-Ho Pyo, Jae Hwan Kim, Youngseon Byeon, Seong Gu Heo, Sun Min Lim, Min Hee Hong, Chang Gon Kim, Hye Ryun Kim, Mi Ran Yun, Byoung Chul Cho. Targeting adaptive metabolic program as a novel treatment approach for TKIs-failed ALK-positive NSCLCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB544.

Abstract 1542: MALAT1 ablation dismantles homologous recombination repair machinery and sensitizes castrate resistant prostate cancer cells to PARP inhibitor

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as a promising target of intervention for metastatic castration-resistant prostate cancer (mCRPC), as approximately one-third of mCRPC patients harbor mutations in the genes associated with the homologous recombination (HR) pathway. Nonetheless, most patients inevitably develop resistance to PARP inhibition (PARPi), due to the induction of reversion mutations in the HR pathway. These mutations restore the function of HR genes and revert to the HR-proficient stage, which in turn decreases the vulnerability of cancer cells to PARP inhibitors. Here, we report that the clinical utility of PARPi could be efficaciously extended by targeting Metastasis associated lung adenocarcinoma transcript (MALAT1), a long non-coding RNA (lncRNA) often elevated in advanced-stage prostate cancer (PCa). In support of this, we show that mCRPC patients exhibit higher expression of genes involved in HR, which positively correlate with MALAT1 levels. Furthermore, RNA interference (RNAi)-mediated depletion of MALAT1 in CRPC cells perturb the expression of key HR genes, namely BRCA1/2, RAD51, EXO1, CHEK1/2, subsequently resulting in HR deficiency. This, in turn, escalates accumulation of DNA lesions in the MALAT1 ablated cells as noticed by abundance in γH2AX, a marker for DNA damage. As a consequence, MALAT1 ablated cells instigates G1/S phase arrest to fetch additional time to repair the damaged DNA or to induce apoptosis. Furthermore, we also provide evidence that the HR deficiency induced by MALAT1 depletion phenocopies “BRCAness” and exhibits synergy with clinically approved DNA repair inhibitors such as Olaparib. In particular, co-inhibition of MALAT1 and PARP1 exhibits a significant anti-proliferative effect reduced clonogenic survival and delays the resolution of γH2AX foci in CRPC cell lines. Taken together, our results establish that MALAT1 plays a central role in regulating the DNA damage response and provides a mechanistic rationale for dual targeting of MALAT1 and PARP in mCRPC patients. Citation Format: Anjali Yadav, Tanay Biswas, Ayush Praveen, Bushra Ateeq. MALAT1 ablation dismantles homologous recombination repair machinery and sensitizes castrate resistant prostate cancer cells to PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1542.

Cancer-associated fibroblasts require proline synthesis by PYCR1 for the deposition of pro-tumorigenic extracellular matrix

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.

KDM6B promotes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key regulator of PARthanatos. Loss of KDM6B protein or its demethylase activity conferred cancer cell resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second XRCC1-dependent repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.