Biology subjects

Shapiro, G. I.

Publications and source records attributed to Shapiro, G. I..

10 recordsLinked to original sources

bioRxiv2026

DNA-PK enforces the S/G2 checkpoint in ATM-deficient cells to limit ATR inhibitor efficacy

ATR inhibition has been proposed as a strategy for tumors under replication stress or those harboring ATM deficiency. However, anti-tumor activity is limited by DNA-PK and ATM compensation. Here, we have delineated distinct roles for DNA-PK and ATM during ATR inhibitor exposure. Both kinases are activated in a cell cycle-dependent manner at the S/G2 boundary following ATR inhibition. DNA-PK imposes the S/G2 checkpoint and promotes completion of DNA replication, whereas ATM detects persistent aberrant DNA replication structures with associated DNA damage in late S and G2/M to establish a G2/M checkpoint promoting DNA repair. In ATM-deficient cells and patient tumors, DNA-PK is activated following ATR inhibition and assumes greater versatility by not only enforcing the S/G2 checkpoint but also by detecting aberrant replication structures in S phase in place of ATM, allowing cells to tolerate ATR inhibition. Consequently, DNA-PK inhibition synergizes with ATR inhibition in ATM-deficient cells and enhances ATR inhibitor efficacy in ATM-deficient PDX models. These data support clinical evaluation of combined ATR and DNA-PK inhibition in ATM-deficient cancers.

cancer biology↗

bioRxiv2026

TALAVE in Breast Cancer: BRCA1/2 Mutation-Dependent Immune Remodeling after PARP Inhibition with Limited Checkpoint Engagement

PARP inhibitors (PARPi) drive efficacy in BRCA-mutant breast cancer (BC) via DNA damage-induced synthetic lethality and immune activation, supporting their combination with immune checkpoint blockade. In the TALAVE study, patients with advanced BRCA-mutant or wild-type (WT) HER2-negative BC received talazoparib followed by talazoparib plus avelumab. Only BRCA-mutant BC responded clinically. Serial multi-omic profiling revealed BRCA-dependent tumor-immune remodeling, including sustained H2AX-pTBK1 signaling with increased CD8+ T cells, tumor cell depletion, and enrichment of CD163+ macrophages. In contrast, BRCA-WT tumors remained compact and immunosuppressed with reduced T cells after therapy. PD-1+ T cells localized to CD4+-rich neighborhoods and correlated with longer progression-free survival in BRCA-mutant tumors. However, PD-L1+ cells were rapidly depleted or confined to immune-excluded regions, spatially segregating them from PD-1+ T cells, impairing effective checkpoint blockade. These findings suggest limited benefit of PD-1/PD-L1 blockade in augmenting PARPi activity and highlight the need for alternative strategies to sustain PARPi-induced immunity.

cancer biology↗

bioRxiv2026

BET BD2 inhibition facilitates SPOP-mediated degradation of chromatin-associated BRD4/BRD4-NUT, a therapeutic vulnerability in NUT carcinoma

BET bromodomain inhibitors block binding of BET family bromodomains 1 and 2 (BD1, BD2) to chromatin and have demonstrated clinical activity in NUT carcinoma (NC), a BRD-NUT fusion-driven cancer, but toxicity from BD1 inhibition has limited their effectiveness. We investigated whether selective inhibition of BRD4 bromodomain 2 (BD2) could retain antitumor activity while reducing toxicity. NC cells were uniquely sensitive to the novel BRD4-BD2 inhibitor DC-9476 and other BD2-selective inhibitors, which induced differentiation and growth arrest. A CRISPR knockout screen identified the BRD4-targeting E3 ligase SPOP as the top resistance hit. BD2 inhibition, but not BD1-selective or pan-BET inhibition, triggered SPOP-dependent proteasomal degradation of BRD4 and BRD4-NUT; SPOP loss prevented degradation and largely rescued BD2 inhibitor-induced differentiation and growth arrest. Unexpectedly, BRD4 and BRD4-NUT remained chromatin-associated during BD2 inhibition, whereas BD1 or pan-BET inhibition displaced them. Together with evidence that ectopic BRD4-NUT expression sensitizes BRD4 to degradation, these findings support a model in which BRD4-NUT megadomains create a high-density, degradation-competent SPOP substrate pool of BRD4 and BRD4-NUT upon BD2 inhibition, whereas pan-BET inhibition disperses this substrate and limits efficient degradation. In preclinical NC models, BD2-selective inhibition achieved greater tumor growth inhibition and survival benefit than pan-BET inhibition, revealing a therapeutic vulnerability.

cancer biology↗

bioRxiv2026

Targeting FGFR signaling overcomes therapeutic resistance and immune evasion in oncogenic PIK3CA-driven serous-like endometrial cancer

Serous endometrial cancer (SEC) is an aggressive subtype of endometrial cancer (EC) with poor prognosis and limited treatment options. Here, we developed a clinically relevant, immunocompetent serous-like mouse model incorporating oncogenic PIK3CA mutation, Trp53 loss, and MYC overexpression. Using this model together with human EC cell lines, patient-derived organoids (PDOs), xenografts, and patient datasets, we investigated mechanisms underlying resistance to PI3K-targeted therapy. Single-cell profiling reveals that FGFR1/2 upregulation associates with intrinsic resistance, whereas FGFR3 characterizes acquired resistance. Dual FGFR and PI3K inhibition produced superior tumor control compared with either agent alone. Mechanistically, FGFR signaling promotes immune evasion by downregulating MHC-I/HLA-mediated antigen presentation and enriching M2-type tumor-associated macrophages. FGFR inhibition reversed these changes and synergized with anti-PD-1 therapy to enhance antitumor immune responses and establish durable immune memory. Collectively, these findings identify FGFR signaling as a key driver of therapeutic resistance and immune escape in SEC and support FGFR-targeted combination strategies.

cancer biology↗

bioRxiv2025

Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

Rhabdoid tumors (RTs) are highly aggressive cancers driven by biallelic mutation of SMARCB1, a core subunit of the BAF (SWI/SNF) complex. We found that SMARCB1-deficient tumors have a defect in the microhomology-mediated end-joining (MMEJ) pathway, and SMARCB1 is essential for maintaining the protein level of the core MMEJ protein Polymerase Q (POLQ). Mechanistically, SMARCB1 facilitates the nuclear export of the POLQ mRNA through its interaction with the nuclear pore complex. Interestingly, loss of MMEJ in RT cells leads to a compensatory activation of, and a hyper-dependence on, the Fanconi Anemia (FA)/BRCA pathway. Knockout or inhibition of this pathway selectively kills RT cells. Notably, RBM39 degraders, novel splicing modulators, effectively inhibit the FA/BRCA pathway and kill RT cells. SMARCB1 and other cBAF/pBAF components are important for maintenance of MMEJ activity and POLQ protein level, suggesting that BAF-deficient cancers more broadly may be treated by targeted inhibition of the FA/BRCA pathway. TeaserSMARCB1-deficient tumors lose POLQ protein and MMEJ repair, forcing a hyper-dependence on the FA/BRCA pathway.

cancer biology↗

bioRxiv2025

Disruption of Microhomology-mediated End-joining in Ewing Sarcoma

Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POL{Theta}, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POL{Theta} expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POL0 mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS. Highlights- Ewing sarcoma tumors have a deficiency in POL{theta} expression and a corresponding loss of MMEJ activity - EWSR1 is a splicing factor that interacts with other splicing factors such as FUBP1 and KHSRP/FUBP2 to accurately splice the POLQ mRNA. - The EWS-FLI1 fusion oncoprotein, or loss of EWSR1, causes a splicing defect, leading to exon25 skipping of the POLQ pre-mRNA and loss of POL{theta} expression - The MMEJ deficiency of EwS cells results in cellular sensitivity to inhibitors of Fanconi Anemia, Homologous Recombination or Non-Homologous End-Joining - Exon 25 skipping of POLQ mRNA is a predictive biomarker for HR inhibitors in human cancers

cancer biology↗

bioRxiv2023

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUTs ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCEIdentification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

cancer biology↗

bioRxiv2021

STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer

Poly (ADP-ribose) polymerase (PARP) inhibitors exert their efficacy by inducing synthetic lethal effects, as well as cGAS/STING-mediated immune responses in BRCA- and other homologous recombination repair-deficient cancer cells. Here we investigated whether the immunologic and therapeutic effects of PARP inhibition in BRCA-deficient breast cancer models could be augmented by synthetic cyclic dinucleotide agonists of STING. Combined PARP inhibition and STING agonism induced a greater degree of STING pathway activation and proinflammatory cytokine production compared to monotherapies in BRCA1-deficient human and mouse triplenegative breast cancer cell lines. In a mouse model of BRCA1-deficient TNBC, the combination also induced an improved immune response compared to either monotherapy alone, evidenced by a greater degree of cytotoxic T cell recruitment and activation, and enhanced dendritic cell activation and antigen presentation. Nanostring mRNA analysis indicated that combinatorial effects were the result of augmented interferon signaling and antigen processing, as well as of heightened leukocyte and dendritic cell functions. Finally, the combination markedly improved anti-tumor efficacy in vivo compared to monotherapy treatment, with evidence of complete tumor clearance and prolongation of survival. These results support the development of combined PARP inhibition and STING agonism in BRCA-associated breast cancer.

cancer biology↗

bioRxiv2020

Polymerase Theta Inhibition Kills Homologous Recombination Deficient Tumors

PARP inhibitors (PARPi) have become a new line of therapy for Homologous Recombination (HR)-deficient cancers. However, resistance to PARPi has emerged as a major clinical problem. DNA polymerase theta (POL{theta}) is synthetic lethal with HR and a druggable target in HR-deficient cancers. Here, we identified the antibiotic Novobiocin (NVB) as a specific POL{theta} inhibitor that selectively kills HR-deficient tumor cells in vitro and in vivo. NVB directly binds to the POL{theta} ATPase domain, inhibits its ATPase activity, and phenocopies POL{theta} depletion. BRCA-deficient tumor cells and those with acquired PARPi resistance are sensitive to NVB in vitro and in vivo. Increased POL{theta} expression levels predict NVB sensitivity. The mechanism of NVB-mediated cell death in PARPi resistant cells is the accumulation of toxic RAD51 foci, which also provides a pharmacodynamic biomarker for NVB response. Our results demonstrate that NVB may be useful alone or in combination with PARPi in treating HR-deficient tumors, including those with acquired PARPi resistance. One Sentence SummaryWe identified Novobiocin as a specific POL{theta} inhibitor that selectively kills naive and PARPi resistance HR-deficient tumors in vitro and in vivo.

cancer biology↗

bioRxiv2019

Reversion and non-reversion mechanisms of resistance to PARP inhibitor or platinum chemotherapy in BRCA1/2-mutant metastatic breast cancer

BackgroundLittle is known about mechanisms of resistance to PARP inhibitors and platinum chemotherapy in patients with metastatic breast cancer and BRCA1/2 mutations. Further investigation of resistance in clinical cohorts may point to strategies to prevent or overcome treatment failure. Patients and MethodsWe obtained tumor biopsies from metastatic breast cancer patients with BRCA1/2 deficiency before and after acquired resistance to PARP inhibitor or platinum chemotherapy. Whole exome sequencing was performed on each tumor, germline DNA, and circulating tumor DNA. Tumors underwent RNA sequencing, and immunohistochemical staining for RAD51 foci on tumor sections was performed for functional assessment of intact homologous recombination. ResultsPre- and post-resistance tumor samples were sequenced from 8 patients (4 with BRCA1 and 4 with BRCA2 mutation; 4 treated with PARP inhibitor and 4 with platinum). Following disease progression on DNA-damaging therapy, four patients (50%) acquired at least one somatic reversion alteration likely to result in functional BRCA1/2 protein detected by tumor or circulating tumor DNA sequencing. Two patients with germline BRCA1 deficiency acquired genomic alterations anticipated to restore homologous recombination through increased DNA end resection: loss of TP53BP1 in one patient and amplification of MRE11A in another. RAD51 foci were acquired post-resistance in all patients with genomic reversion, consistent with reconstitution of homologous recombination. All patients whose tumors demonstrated RAD51 foci post-resistance were intrinsically resistant to subsequent lines of DNA-damaging therapy. ConclusionsGenomic reversion in BRCA1/2 was the most commonly observed mechanism of resistance, occurring in 4 of 8 patients. Novel sequence alterations leading to increased DNA end resection were seen in two patients, and may be targetable for therapeutic benefit. The presence of RAD51 foci by immunohistochemistry was consistent with BRCA1/2 protein functional status from genomic data and predicted response to later DNA-damaging therapy, supporting RAD51 focus formation as a clinically useful biomarker.

cancer biology↗