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Rare, recurrent balanced translocations occur in a variety of cancers but are often not functionally interrogated. Balanced translocations with the immunoglobulin heavy chain locus (IGH; 14q32) in chronic lymphocytic leukemia (CLL) are infrequent but have led to the discovery of pathogenic genes including CCND1, BCL2, and BCL3. Following identification of a t(X;14)(q28;q32) translocation that placed the mature T cell proliferation 1 gene (MTCP1) adjacent to the immunoglobulin locus in a CLL patient, we hypothesized that this gene may have previously unrecognized importance. Indeed, here we report overexpression of human MTCP1 restricted to the B cell compartment in mice produces a clonal CD5+/CD19+ leukemia recapitulating the major characteristics of human CLL and demonstrates favorable response to therapeutic intervention with ibrutinib. We reinforce the importance of genetic interrogation of rare, recurrent balanced translocations to identify cancer driving genes via the story of MTCP1 as a contributor to CLL pathogenesis., Some genes that are part of balanced translocations are reported as drivers for tumourigenesis. Here, the authors report a translocation involving MTCP1 in chronic lymphocytic leukemia and show that MTCP1 overexpression leads to the disease in a murine model.

Background: Chronic Lymphocytic Leukemia (CLL) is a genetically heterogeneous disease characterized by clonal expansion of B-lymphocytes that induce secondary immune suppression. CLL is now treated with inhibitors of Bruton tyrosine kinase (BTK) and BCL2. Virtually all patients respond to therapy, however resistance to these therapies has been described justifying the need for novel CLL therapies. Broad inhibition of cyclin dependent kinases (CDK) and associated alternative target enzymes with agents such as flavopiridol or dinaciclib have demonstrated significant clinical activity in CLL but are hindered by a relatively narrow therapeutic window. VIP152 is a highly specific inhibitor of CDK9 - considered the most important CDK kinase member for CLL clinical activity. VIP152 has favorable pharmacokinetic properties and has demonstrated durable, preliminary single-agent clinical activity in double-hit diffuse large B-cell lymphoma. Herein, we report the efficacy of VIP152 preclinically in CLL. Methods: On-target activity of VIP152 was measured using a KinomeScan from DiscoveryRx at 100nM and 1000nM. Kinase profiling for VIP152 was performed on 6 kinases in a 10-dose assay at ReactionBio. Cell-based viability and proliferation assays (MTS, Annexin-V (AV), and propidium iodine (PI)) were performed in primary CLL cells and the CLL cell lines, HG3 and MEC1. Transcriptional activity after VIP152 exposure was measured via qPCR and limiting-cell RNA sequencing (lcRNAseq). Proteomic and immunoblot studies were performed to measure perturbations in CDK9 binding partners and on-target activity of VIP152. A genome-wide CRISPR/CAS9 knockout screen was performed to identify any synthetically lethal targets and pathways. Results: The KinomeScan identified CDK9 as the kinase with maximal inhibition upon VIP152 treatment and no other CDKs were identified at 100nM. Kinase profiling revealed the IC 50 of VIP152 was lowest for CDK9/Cyclin T1 and CDK9/Cyclin T2 with close similarity to dinaciclib and greater than 1 log superiority over KB-0742. Co-immunoprecipitation and proteomics experiments have identified a CDK9 specific mechanism of action relating to perturbations of CDK9 binding partners. Specifically, we showed that CDK9 nuclear immunoprecipitation resulted in decreased co-immunoprecipitation of 7SK RNA components (HEXIM1 & MEPCE) as well as decreased RNA Polymerase II (RNAP2). The decrease in RNAP2 CoIP was further seen via proteomics. A 2-hour exposure of VIP152 against HG3 and MEC1 demonstrated growth inhibition, with an IC 50 of 0.9814µM and 1.092µM respectively. Continuous exposure of the compound for 24 hours resulted in a statistically significant drop in relative viability of 30% across a 10-fold dose range (0.1µM to 1.0µM) as measured by AV/PI. Primary CLL cells (n=10) responded with similar dosing strategies with a 54% reduction in viability at 1µM; moreover, stromal cell co-culture experiments demonstrated VIP152's ability to induce cell death and overcome stromal protection with short exposure. Induction of apoptosis was observed with pro-caspase-3 and PARP cleavage on immunoblot. qPCR and immunoblot studies demonstrated a time dependence of phosphorylated serine 2 (pS2) RNAP2 decreases alongside diminishment of MYC and MCL1 mRNA and protein. pS2 was shown to decrease as early as 2 hours after VIP152 treatment with similar decreases in MCL1 and MYC at both the protein and mRNA levels. We identified several pathways which are disrupted following treatment via lcRNAseq, including TNFR1 and TNFR2 signaling as well as upregulation of autophagy signals. Finally, CRISPR screen identified several potentially synergistically lethal targets, including transcriptional co-activators, DNA binding proteins, and cell proliferation pathways. Validation of these is ongoing as is an in vivo study of VIP152 in a CLL mouse model. Conclusions: Our data demonstrate VIP152 to be a highly selective and potent CDK9 inhibitor that disrupts the CDK9 nuclear complex and mediates significant preclinical activity against CLL cell lines and primary CLL cells. VIP152 also demonstrates predictable and new pharmacodynamic markers to assess target engagement. Collectively, these data support the recently initiated CLL clinical trial (NCT04978779). Disclosures Johnson: Vincerx: Current Employment; Janssen: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Frigault: Vincerx Pharma Inc: Current Employment; AstraZeneca: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months, Patents & Royalties. Greer: Gilead: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Vincerx Pharma Inc: Current Employment. Hamdy: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties. Izumi: Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Hwang: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees.

Introduction & Objectives: Efforts to characterize the heterogeneity of advanced hematologic malignancies using large-scale genomic studies have identified recurrent monoallelic mutations affecting the E571 residue of the essential nuclear exporter, Exportin-1 (XPO1; E571K in ~80% of cases, E571G in ~15% of cases). E571-XPO1 mutations alter the charge and structural basis of the cargo-binding region, disrupting critical biophysical interactions between XPO1 and its' cargos. Enriched in hematologic malignancies, E571-XPO1 mutations are predominantly reported in chronic lymphocytic leukemia (CLL; 5-10% of cases), classical Hodgkin's lymphoma (~25% of cases), and primary mediastinal B cell lymphoma (PMBCL; 25-30% of cases). The subsequent change in XPO1-cargo localization alters the transcriptional profile and overall phenotype of the leukemic cell, with evidence suggesting hyper-active NF-κB and NFAT signaling pathways as leading leukemogenic mechanisms. Moreover, while overall immune dysfunction in CLL leads to infections as a major cause of morbidity and mortality, CLL patients with E571-XPO1 mutations are more susceptible to death by infection, suggesting these mutations may exacerbate the leukemia-induced immunosuppressive phenotype. Similarly, inactivating mutations/deletions to A20 (TNFAIP3 gene), the master regulator of NF-κB, are recurrently reported in several B cell malignancies but most frequently observed in PMBCL (~30% of cases). E571-XPO1 mutations and TNFAIP3 deletions/mutations have been found as co-occurring genetic abnormalities in PMBCL, and while TNFAIP3 mutations in CLL are rare, functional convergence on NF-κB and immune signaling suggests altered XPO1 and A20 activity may have unreported pathogenic significance in CLL. Thus, we aimed to explore the oncogenic and immunologic consequence of co-occurring XPO1 and A20 abnormalities by evaluating a novel in-vivo model recapitulating this scenario. Methods: To explore concurrent aberrations to XPO1 and A20, we developed a novel mouse model to recapitulate this event (Eµ-XPO1xA20 KO). This model was generated by crossing the Eµ-XPO1 transgenic mouse - which overexpresses wildtype (WT), E571K, or E571G-XPO1 under control of a VH promoter-IgH-Eµ enhancer to target transgene expression to immature and mature B cells - with a B cell-specific A20 inactivation mouse (A20 KO) - which lacks functional A20 as a result of Cre recombinase-mediated excision of TNFAIP3 exon 3 via loxP recombination sites flanking this region and Cre recombinase expressed under CD19 promoter/enhancer elements. Eµ-XPO1 and Eµ-XPO1xA20 KO mice were aged and followed, and their B and T cell repertoire was assessed via flow cytometry. Results & Conclusion: We previously demonstrated Eµ-XPO1 mice develop a CLL-like disease (CD19+/CD5+/B220dim B lymphocytes), but leukemia development is significantly delayed - evident between 20-30 months of age. Preliminary analysis in adolescent animals revealed irregular lymphocyte populations as early as 6 months of age in the blood and spleen of Eµ-XPO1xA20 KO mice when compared to non-transgenic and Eµ-XPO1 mice; highlighted by elevated populations of CD93+/CD23+ transitional B cells and CD3+ T cells, and reduced populations of CD21+/IgM+ marginal zone B cells. Moreover, development of a circulating CLL-like disease accompanied by palpable lymphadenopathy and splenomegaly was observed in Eµ-XPO1xA20 KO mice as early as 17-20 months of age, again presenting a distinct immunophenotype inconsistent with that observed in Eµ-XPO1 mice. Additionally, progressive accumulation of CD3+/CD19- T cell leukemia-like populations were observed in a subset of Eµ-XPO1xA20 KO and A20 KO mice, indicating these aberrations may further disrupt and stimulate uncontrolled proliferation affecting the overall immune repertoire. Significance: We report that simultaneous disruption of essential regulators XPO1 and A20 in murine B cells encourages development of irregular B and T cell populations, and can stimulate a progressive CLL-like or T cell leukemia-like expansion. Continued investigation with these models can further our understanding of the relationship between overall immune function and these critical regulatory molecules, and can provide considerable insight to identifying pathways for selective targeting as a personalized therapy in several high-risk cancer types. Disclosures Byrd: AstraZeneca: Consultancy; Takada: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Syndex: Consultancy; Trillium: Consultancy; Vincera Pharmaceuticals: Current equity holder in publicly-traded company.

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in Western countries and is spelled by substantial genetic and clinical heterogeneity. During CLL transformation, loss or gain of genetic material appears to be a key determinant of disease phenotype and clinical outcome, with major chromosome aberrations observed in up to 80% of patients. Alternatively, balanced translocations, specifically those resulting in constitutive over-expression of various proto-oncogenes under the immunoglobulin heavy chain locus (IGH; 14q32), occur far less frequently. Despite their infrequence, molecular profiling of these rare rearrangements have revealed broad importance of un-recognized genes critical to the pathogenesis of CLL. Employing this strategy, we identified a young CLL patient with a previously undescribed t(X;14)(q28;q32) translocation, co-localization of the mature T cell proliferation 1 (MTCP1; Xq28) coding region with the IGH locus, triggering overexpression of MTCP1 in the CLL cells. Translocations involving MTCP1 are a driving factor in T-prolymphocytic leukemia; however, a role for MTCP1 in CLL has not been described. Inspired by this observation, we screened >1700 suspected CLL cases and evaluated gene expression data for further evidence of MCTP1 aberrations. This query identified seven additional Xq28 rearrangements, revealed MTCP1 mRNA was globally over-expressed in CLL cells compared to normal B-cells, and increased MTCP1 mRNA expression portends a poor response to chemoimmunotherapy. To establish a role for MTCP1 as an oncogene in B cell malignancies, we generated a mouse model with B cell-specific MTCP1 overexpression (Eµ-MTCP1). Longitudinal evaluation revealed a majority of Eµ-MTCP1 mice developed a lethal hematologic malignancy between 5-12 months of age, highlighted by the progressive emergence of clonally related CLL-like B lymphocytes (CD19+/CD5+ B cells) in the blood and accumulating in the spleen and lymph nodes. To support the use of the newly generated Eµ-MTCP1 mouse as a tool for pre-clinical evaluation of CLL therapeutics, we demonstrate that continuous ibrutinib administration in Eµ-MTCP1 mice was sufficient to delay the onset of the CLL-like disease and significantly prolonged survival. In summary, we report Xq28 translocations as rare genetic abnormalities in CLL, yet being one mechanism by which CLL cells amplify expression of MTCP1 compared to normal B cell subsets. Further, the Eµ-MTCP1 mouse model should be considered as an alternative tool for both biologic assessment of co-expressed genes and pre-clinical evaluation of novel CLL therapeutics. Lastly, relevant to all cancer types, successful application of a strategy pursuing the functional consequence of genes involved in rare translocations contributed to the understanding of this disease and identified a novel target for future therapeutic consideration. Citation Format: Janek S. Walker, Zachary A. Hing, Steven Sher, James Cronin, Katie Williams, Bonnie Harrington, Jordan N. Skinner, Casey B. Cempre, Charles T. Gregory, Max Yano, Larry P. Beaver, Brandi R. Walker, Jadwiga M. Labanowska, Nyla A. Heerema, Krzysztof Mrozek, Jennifer A. Woyach, Amy S. Ruppert, Amy Lehman, Hatice Gulcin Ozer, Vincenzo Coppola, John C. Byrd, James S. Blachly, Rosa Lapalombella. Evaluating a rare t(X;14)(q28;q32) translocation reveals MTCP1 as a driving factor in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2260.