Gwin, Kimberly

Abstract Introduction: Large B-cell lymphoma (LBCL) harboring chromosomal rearrangements (double-hit, DH) or coexpression of MYC and BCL2 (double-expressor, DE) represents biologically aggressive subgroups with inferior clinical outcomes. Oncogene-driven DNA replication stress (ODRS) is a hallmark of MYC and BCL2 dysregulation; however, the DNA damage response (DDR) pathways that sustain replication and cell survival under ODRS remain incompletely defined in DLBCL. Identifying DDR dependencies induced by ODRS may reveal therapeutically actionable vulnerabilities in DH/DE-LBCL. Methods: We studied DH/DE-LBCL cell lines (OCILY1, SUDHL2, DOHH2), non-DH/DE-LBCL cell lines (HT, KARPAS422, HBL1), and benign retinal pigment epithelial (RPE) cells engineered to overexpress MYC and/or BCL2. ODRS and DNA damage were assessed by phosphorylation of RPA (S4/S8, S33) and H2AX histone (γH2AX), respectively, using immunofluorescence, immunohistochemistry, and western blot (WB). Activity of homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ) pathways were quantified with established reporter assays. POLΘ (gene: POLQ) expression, an MMEJ marker, was evaluated by qPCR and WB. DDR activity was assessed using genomic signatures from whole-exome sequencing data of 404 LBCL patients. In vitro sensitivity of LBCL cells to POLΘ and PARP inhibitors was assessed with publicly available small-molecule inhibitors using CellTiterGlo and clonogenic assays, and therapeutic efficacy was validated in a DH-LBCL xenograft model in NRG mice. Results: DH/DE-LBCL cells and primary tumors exhibited increased ODRS and persistent DNA damage compared with non-DH/DE-LBCL models, including accumulation of γH2AX in mitosis. Functional profiling revealed selective upregulation of MMEJ in MYC/BCL2-overexpressing cells, with minimal changes in HR or NHEJ activity. POLQ expression was significantly increased in DH/DE-LBCL cell lines and primary samples, and the ID6 signature (associated with MMEJ activity) was significantly enriched in DH-LBCL tumors compared to non-DH/DE-LBCL tumors. POLΘ inhibition demonstrated greater toxicity in DH/DE-LBCL and in MYC-overexpressing RPE cells compared to non-DH/DE-LBCL and wild-type RPEs. Combined inhibition of POLΘ and PARP resulted in marked synergy in vitro. The therapeutic efficacy of POLΘ inhibition, with the synthetic lethality of concurrent PARP inhibition, was validated in the DH-LBCL xenograft model. Concurrent POLΘ and PARP inhibition with small-molecule inhibitors induced an antitumor effect in vivo, with increased overall survival in mice compared with vehicle- or olaparib-single-agent-treated groups. Conclusions: ODRS in DH/DE-LBCL creates a selective dependence on MMEJ mediated by POLΘ. Therapeutic targeting of POLΘ, alone or in combination with PARP inhibition, exploits this vulnerability and represents a promising strategy for this high-risk LBCL subset. These findings provide a biological rationale for clinical translation of POLΘ-directed therapies in DH/DE-LBCL. Citation Format: Cecilia Ayala-Zambrano, Dongni Yi, Michelle Manske, Kimberly Gwin, Richar Kandasamy, Surendra Dasari, Anne Novak, Shuhei Asada, Xiaosheng Wu, Zhenkun Lou, Thomas Witzig, Jithma Abeykoon. c-MYC/BCL2 induced replication stress drives dependence on POLQ-mediated microhomology-mediated end joining in aggressive large B-cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A029.

Abstract Description Hematopoiesis declines with age due to intrinsic and extrinsic dysregulation resulting in diminished lymphopoiesis. Hoxa9 is a pleiotropic hematopoietic regulator necessary for stem/progenitor cell (HSPC) maintenance and lymphoid differentiation. Interestingly, bone marrow (BM) HSPC and B lineage compartments of young hoxa9-/- mice mimic that which accompanies aging. Hoxa9 is a negative regulator of p16ink4a, a cyclin-dependent kinase inhibitor upregulated in hoxa9-/- HSPCs and a gatekeeper of the G1 to S cell cycle checkpoint. We hypothesized that increased expression of p16ink4a contributes to the HSPC and/or B progenitor alterations due to hoxa9-deficiency. Interestingly, BM B lymphopoiesis was normalized in hoxa9-/-p16ink4a-/- (DKO) mice, but alterations were less obvious in HSPCs. Flow analysis of the HSPC compartment proliferation showed contraction of unbiased but not myeloid-biased progenitors prior to lineage fate commitment. hoxa9-/- CFU-PreB were blunted then rescued by p16ink4a co-ablation. Primary BM transplant demonstrates DKO like hoxa9-/- has reduced reconstitution efficiency but differs with superior B lineage reconstitution similar to controls. Bioinformatic analysis of recent open access CUT&RUN data in HSPCs confirms Hoxa9 binds promoters in genes relevant to G1 to S cell cycle checkpoint, these findings currently being validated. Our work emphasizes HSPC regulatory factors as crucial targets in combating age-associated maladies of the immune system. Funding Sources Supported by NIH R01AG076163 Topic Categories Hematopoiesis and Immune System Development (HEM)

Oncogene expression can cause replication stress (RS), leading to DNA double-strand breaks (DSBs) that require repair through pathways such as homologous recombination, nonhomologous end-joining, and microhomology-mediated end-joining (MMEJ). Cyclin D1 (encoded by CCND1 ) is a well-known oncoprotein overexpressed in cancer; however, its role in RS is unknown. Using mantle cell lymphoma (MCL) as a naturally occurring model of cyclin D1 overexpression, we examined the impact of cyclin D1 on RS and DSB repair mechanisms. Cyclin D1 overexpression elevated RS, increased DNA damage, especially during mitosis, and caused specific upregulation of MMEJ. Furthermore, cyclin D1 activated polymerase theta ( POLQ ) transcription by binding its promoter loci, driving POLΘ-mediated MMEJ that is essential to withstand cyclin D1–induced RS. Moreover, concurrent ATM deficiency further intensified RS, enhanced POLQ expression, and heightened reliance on MMEJ-mediated DNA damage repair. Consequently, inhibition of POLΘ in cyclin D1–overexpressed settings further exacerbated RS, causing single-strand DNA gap accumulations and chromosomal instability, ultimately leading to apoptosis, an effect amplified in ATM-deficient cells. Targeting MMEJ via POLΘ inhibition is therefore an effective strategy in the context of cyclin D1 overexpression and ATM deficiency and may provide a unique therapeutic approach for treating MCL and other malignancies characterized by similar alterations.

Oncoprotein-driven human cancers exhibit replication stress (RS), leading to an increase in DNA double-strand breaks (DSB). These DSBs are repaired by one of the three main pathways - homologous recombination repair (HR), non-homologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ). Cyclin D1 (CCND1) is one of the most overexpressed oncoproteins and is universally overexpressed in mantle cell lymphoma (MCL). While CCND1 overexpression drives cells into the S-phase, its specific effects on RS and DSB repair pathways are poorly understood. Here, we investigated the impact of CCND1 overexpression on RS and DSB repair mechanisms in MCL. First, the expression level of CCND1 positively correlated with heightened RS, as indicated by elevated p-RPA-S33 and p-RPA S8 levels, and increased DNA damage, assessed by g-H2AX, especially in the mitotic phase, across diverse cell models, including MCL. Since CCND1-overexpressed cells proliferate rapidly, we hypothesized that some DNA repair pathways might be upregulated to meet the demand for repairing the excess RS-driven DSBs. Reporter assays demonstrated that the MMEJ pathway was specifically upregulated upon CCND1 overexpression, whereas no significant changes were observed in the HR and NHEJ pathways. Furthermore, CRISPR interference (CRISPRi)-mediated knockdown of CCND1 in MCL cells led to a significant reduction in MMEJ activity. This finding suggests the specificity of CCND1 in mediating the upregulation of the MMEJ pathway. Consistent with this, increased MMEJ activity, assessed via foci formation and immunoblotting of DNA polymerase theta expression (POLQ, encoded by POLQ gene), the primary polymerase for the MMEJ pathway, correlated with CCND1 overexpression, suggesting the crucial role of MMEJ in DSB repair processes under CCND1-overexpressed conditions. Sincethe MMEJ pathwayplays a pivotal role in repairing DSB during mitosis, we assessed the dependency of MCL cells on MMEJ-mediated DSB repair for cell survival. As expected, genetic deletion or pharmacological inhibition (ART558 and novobiocin) of POLQ in MCL cells led to increased RS and increased unrepaired DNA damage during mitosis, ultimately leading to chromosomal instability, mitotic catastrophe, and apoptosis. Additionally, the deficiency of POLQ in MCL cells resulted in increased levels of single-strand DNA, another critical biomarker for RS. Second, we found that ATM deficiency, the second most common genetic alteration after CCND1 overexpression in MCL, further increased the cellular dependence on MMEJ. Interestingly, the accumulation of unrepaired DNA damage during mitosis was notably increased in ATM-deficient CCND1-overexpressed cells. When POLQ was inhibited, a significant antitumor effect was evident in MCL cells, including ibrutinib-resistant MCL, both in vitro and in vivo, and this effect was enhanced by ATM deficiency. Further, simultaneous inhibition of both ATM (AZD0156) and POLQ was synthetic lethal in MCL cell lines and primary tumor cells from patients with MCL. Mechanistically, we observed the direct binding of CCND1 to the POLQ promoter and a corresponding upregulation of POLQ mRNA in CCND1-overexpressing cells. Consistently, a significant overexpression of POLQ mRNA was observed in primary MCL cells, compared to other non-Hodgkin lymphoma cells without CCND1 overexpression. Moreover, the positive correlation between POLQ and CCND1 expression was further validated using syngeneic B-lymphoma cells with and without CCND1 overexpression. Importantly, CRISPRi-mediated CCND1 knockdown reduced POLQ mRNA expression and the dependency of MCL cells on the MMEJ pathway for survival. In addition, ATM deficiency showed a further increase of endogenous POLQ expression and foci formation in cells with CCND1 overexpression, suggesting ATM negatively regulates the MMEJ pathway, especially in the CCND1 overexpressed background. In summary, CCND1 overexpression leads to a substantial increase in RS, intensifying the reliance on MMEJ-mediated mitotic DSB repair. Within MCL, POLQ is crucial in mitigating the adversities of CCND1-driven RS. Our data suggest that targeting the MMEJ pathway via POLQ inhibition, particularly in the presence of CCND1 overexpression and ATM deficiency, emerges as a promising therapeutic approach in oncoprotein-driven hematologic malignancies such as MCL.

Abstract The homeo-domain transcription factor Hoxa9 is required for normal B and T lymphocyte development in bone marrow (BM) and thymus, respectively. Here we show that Hoxa9-deficiency is accompanied by increased expression of the cdkn2a tumor suppressor locus that encodes the cell cycle inhibitor p16Ink4a. Furthermore, we found that somatic deletion of p16Ink4a is sufficient to rescue the B and T cell progenitor deficiencies, as well as the BM lymphoid progenitor pools, in hoxa9−/− mice. In contrast, the hematopoietic stem cell (HSC) and multipotential progenitor (MPP) abnormalities characteristic of Hoxa9-deficient mice were not corrected by loss of p16Ink4a. These new data reveal that Hoxa9 repression of p16ink4a is essential for lymphoid lineage development in BM and thymus. Importantly, they highlight distinct functions for Hoxa9 in regulation of primitive hematopoietic progenitors versus those committed to the B or T cell fates.

Abstract The consequences of immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The known immune abnormalities are not able to consistently explain these complications and it is not easy to correct their immune status. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced functional capacity of hematopoietic progenitors in BM using colony forming unit assays and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations. Consistent with the reduction in HSPCs, monocytes and natural killer cells in BM were reduced and the reduction in BM was recapitulated in blood. Finally, we report imbalances in protein levels of the transcriptional regulators HIF-1α, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients, adding to the complex immunodeficiency state of these patients and the reduced capacity of the innate immune system.

Abstract The generation of B lineage lymphocytes in bone marrow is contingent upon the combinatorial activities of transcription factors, signaling molecules, chromatin modifiers, and microRNAs. One transcription factor important for the generation of B cell precursors is the homeodomain protein HoxA9. Equally important as HoxA9 function in facilitating lymphoid lineage specification is silencing HoxA9 activity upon commitment to the B cell fate as sustained expression impairs later stage B cell differentiation. We previously showed that transcripts for the B cell specification factor EBF1 show an inverse expression pattern to HoxA9. Furthermore, EBF1 does not directly suppress HoxA9 transcription. Hox proteins are regulated by microRNAs in some developmental contexts. MicroRNA profiling of an EBF1-/- cell line stably expressing an inducible EBF1:ER fusion protein revealed a single miR induced by EBF1 activity, miR-204. Importantly, forced expression of miR-204 in EBF1-/- cells repressed transcription and protein levels of the HoxA9 co-factor, Meis1. We hypothesized that if Meis1 is a critical target of miR-204, then sustained expression of miR-204 should phenocopy conditional deletion of Meis1 or HoxA9. Indeed, we determined that forced expression of miR-204 in wildtype multipotent progenitors impaired B cell differentiation, similar to loss of HoxA9. These data suggest a novel role for EBF1 in B cell development, induction of miR-204 to silence a HoxA9-Meis1 transcriptional axis.

Abstract The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.

Abstract Evidence is accumulating that the molecular circuitry initiating lymphoid lineage specification correlates with expression of the receptor tyrosine kinase Flt3. Recombination activating gene-1 (RAG1) locus activation is a hallmark of lymphoid lineage specification. Previous studies established that expression of a RAG1-GFP/+ reporter begins in a subset of lineage negative (Lin-) cells that express high levels of c-kit and Sca-1 (LSK+). To investigate a regulatory connection between Flt3 signaling and RAG1 locus activation from this stage, RAG1-GFP/+ knockin mice expressing wildtype, heterozygote, and knockout Flt3-ligand (FL) alleles were generated. Mono-allelic expression of the FL gene reduced FL transcripts by half, providing an in vivo model to evaluate threshold requirements for FL in lymphocyte development. Percentages and numbers of LSK+ cells were similar in FL+/+ and FL+/- mice, but significantly decreased in FL-/- animals. Analysis of GFP expression within the LSK+ compartment revealed a FL dose dependent reduction in frequencies and numbers of GFP+ cells. The requirement for threshold levels of FL in regulation of GFP expression extended into the B220- c-kitlo IL-7R+ lymphoid progenitor and CD43+ B220+ Pro-B compartments, but not in later stage B cell precursors. These findings establish a critical requirement for FL in regulating one event in lymphoid specification, RAG1 locus activation. These studies were supported by startup funds to K.L.M. from Mayo Clinic.