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Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR-Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncover a selective dependency on RNA splicing factors whose loss preferentially enhances response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augments response to venetoclax in leukemia yet is completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition leads to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. Inhibition of splicing kinase families CLKs (CDC-like kinases) and DYRKs (dual-specificity tyrosine-regulated kinases) leads to aberrant splicing of key splicing and apoptotic factors that synergize with venetoclax, and overcomes resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments.

Background Low‐molecular‐weight heparin has been the preferred treatment of cancer‐associated thrombosis (CAT); however, emerging data support the use of direct oral anticoagulants (DOACs). Objectives The Memorial Sloan Kettering Cancer Center Clinical Pathway has served as the institutional guideline for the use of rivaroxaban to treat CAT since 2014. Key elements are to recommend against use of a DOAC in patients with active gastrointestinal (GI) or genitourinary tract lesions, and a prespecified dose reduction in the elderly (75+ years old). We present our institutional experience for treatment of CAT. Methods From January 2014 through September 2016, 1072 patients began rivaroxaban treatment for CAT; 91.9% had a solid tumor, 8.1% had hematologic malignancies, and 75% of patients with solid tumors had metastatic disease. All patients with CAT treated with rivaroxaban were included in this analysis, regardless of adherence to the Clinical Pathway. Results The 6‐month cumulative incidence of recurrent venous thromboembolism and major bleeding were 4.2% (95% confidence interval [CI], 2.7%‐5.7%) and 2.2% (95% CI, 1.1%‐3.2%), respectively. The incidence of clinically relevant non–major bleeding leading to discontinuation of rivaroxaban for at least 7 days was 5.5% (95% CI, 3.7%‐7.1%), and 73.3% of major bleeds occurred in the GI tract. The 6‐month cumulative mortality rate was 22.2% (95% CI, 19.4%‐24.9%). The elderly had similar rates of recurrent thrombosis and bleeding as those aged under 75 years. Conclusion Our institutional experience suggests that in appropriately selected patients, rivaroxaban may be used for treatment of CAT with promising safety and efficacy.

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Recently, the protein LZTR1 (leucine zipper-like transcriptional regulator 1) was discovered as an adaptor for a cullin 3 complex responsible for ubiquitin-mediated degradation of RAS proteins. While these data provided a novel mechanism for RAS protein regulation, there is considerable controversy as to which RAS paralogs are physiologic substrates of LZTR1. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations in both LZTR1 as well as the RAS GTPase and LZTR1 substrate RIT1 were identified in patients with clonal hematopoietic disorders. However, the effects of these alterations on normal and maliganant hematopoiesis have not been evaluated. Here we utilized a series of genetically engineered murine models for germline and conditional deletion of LZTR1, RIT1, and expression of oncogenic RIT1 mutant which revealed a key role for LZTR1 in the regulation of hematopoietic stem cell (HSC) self-renewal and delineated a series of LZTR1-regulated substrates in hematopoietic cells. Consistent with a role for LZTR1 alterations in the Noonan Syndrome, germline homozygous deletion of Lztr1 was associated with lethality between embryonic day 17.5 and birth. Lztr1-/- fetuses had massive dyserythropoiesis and apoptosis of fetal liver hematopoietic cells. Competitive transplantation of E14.5 Lztr1 null fetal liver or bone marrow from 6-week-old Mx1-cre Lztr1 conditional knockout (cKO) mice resulted in striking increased self-renewal in primary and secondary competitive transplantation assays in vivo (Fig.A-B). Interestingly, recipient animals reconstituted with Lztr1-/- cells developed fatal myeloid and lymphoid leukemias characterized by anemia, thrombocytopenia, and increased myeloid and B-lymphoid cells (Fig.C-D). In order to identify the LZTR1 substrates responsible for effects on HSCs, we evaluated levels of all RAS GTPases in Lztr1 null HSCs. This revealed elevated KRas, NRas, MRas, and Rit1 protein in LZTR1 KO cells (Fig.E), with RIT1 being most prominently elevated. Evaluation of a cohort of 4,113 patients with hematologic malignancies identified 41 patients with somatic RIT1 mutations, the majority of which cluster in the switch II region and escape LZTR1-mediated ubiquitination, resulting in RIT1 protein accumulation (Fig.F-H). Given that the impact of RIT1 mutations on hematopoiesis is unknown, we next compared Lztr1 cKO with conditional expression of one of the most common leukemia-associated RIT1 mutants that escapes LZTR1-mediated ubiquitin (Rit1 M90I). Both Lztr1 cKO and Rit1 M90I conditional expression conferred GM-CSF hypersensitivity to HSCs in vitro, cytokine independent growth to human AML cell lines in vitro, and strong competitive self-renewal in vivo (Fig. I-J). Consistent with RIT1 mutations being found primarily in myeloid neoplasm patients, aged Mx1-cre Rit1M90I/WT mice developed fatal MPN, MDS, and mixed MDS/MPN disorders (Fig.K), which were serially transplantable into sublethally irradiated recipients. Despite convergent effects of LZTR1 and RIT1 on clonal HSC advantage, LZTR1 null cell lines did not solely require RIT1 for HSC advantage as revealed by Lztr1/Rit1 double KO mice. We therefore next carried out a series of experiments in RAS-less cells and whole genome CRISPR screens to delineate factors required for LZTR1 mediated hematopoietic transformation. This revealed that KRAS as well as MRAS and its RAF phosphatase partner SHOC2 were selective dependencies for LZTR1-mediated transformation. These data indicate that multiple RAS GTPases as well as RAF activation are required for LZTR1-mediated transformation (Fig.L). While considerable prior research has evaluated oncogenic alleles of RAS which alter RAS-GTP hydrolysis on hematopoiesis, the role of modulating RAS protein abundance on hematopoiesis is unknown. Here we identify RAS proteolysis as a novel regulator of HSC function, define the spectrum of RIT1 mutations in leukemia, and identify LZTR1 and RIT1 mutations as drivers of leukemogenesis. The discovery of RAS proteolysis as a novel driver of leukemogenesis has important therapeutic implications given efforts to therapeutically degrade RAS family members. Finally, the clinical importance of K/NRAS mutations on resistance to therapies in AML motivates future studies on the potential clinical impact of LZTR1 and RIT1 alterations in myeloid neoplasm patients. Figure 1 Figure 1. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Resistance to therapy is one of the most significant challenges in the treatment of acute myeloid leukemia (AML). While great efforts have uncovered genetic mechanisms of resistance to certain AML-directed therapies, to date, treatment resistance in AML has only partially explained by acquired genetic alterations. Here, we performed genome-wide CRISPR/Cas9 screens to identify drug-gene interactions that modulate therapeutic response to treatments commonly used in AML. Interestingly, our findings uncovered several genes that regulate pre-mRNA splicing whose loss strongly synergized with venetoclax, a BH3 mimetic that blocks the antiapoptotic protein BCL-2. To further delineate the role of RNA processing in response to AML treatments, we performed secondary CRISPR screens with a domain-focused gRNA library targeting 490 RNA processing factors in the presence of various AML drugs. Overall, these genetic screens identified a number of RNA splicing factors whose loss-of-function sensitized AML cells to BCL2 inhibition (Fig. A). Among the top gene candidates whose loss promoted venetoclax efficacy was the splicing factor RBM10 (Fig.B). Strikingly, loss of RBM10 exclusively synergized with venetoclax-based treatments across AML therapeutics, including in TP53 mutant lines (Fig.C-D). Moreover, RBM10 loss restored venetoclax sensitivity to AML cell line variants with acquired venetoclax resistance. Interestingly, while many RNA splicing factors are pan-essential, generation of an Rbm10 conditional knockout mouse revealed that Rbm10 is completely dispensable for steady-state normal hematopoiesis (Fig.E). Since RBM10 has not been studied previously in hematopoiesis, we mapped the impact of RBM10 on mRNA expression and splicing using RNA-seq and direct RNA binding partners genome-wide by eCLIP-Seq (Fig. F). RBM10 loss was strongly associated with downregulation of BCL2A1, an anti-apoptotic factor whose expression is correlated with venetoclax resistance in AML (Fig.G-H). This was dependent on RBM10's ability to bind RNA and expression of BCL2A1 cDNA fully rescued the growth-inhibitory effect of RBM10 KO-venetoclax treated AML cells. Overall, the above data support RBM10 as a synthetic lethal vulnerability in venetoclax therapy. Beyond RBM10, our genetic screens also identified several splicing factors belonging to the family of serine and arginine-rich (SR) proteins whose loss synergized with venetoclax treatment (Fig. I). SR proteins are essential for pre-mRNA splicing and are substrates for phosphorylation by conserved family of kinases, such as Cdc2-like kinases (CLKs) and (dual-specificity tyrosine-regulated kinases) DYRKs. We therefore utilized a series of selective pan-CLK/DYRK1A inhibitors, including SM09419 and SM08502, that potently suppress SR protein phosphorylation. Interestingly, BCL2 is one of the top genetic dependencies upon DYRK1A genetic suppression in prior work from the DepMap (Fig. J). Pharmacologic inhibition of CLK/DYRK1A exhibited high in vitro efficacy at nanomolar range across a diverse range of AML subtypes including cell lines with acquired venetoclax resistance (Fig.K). Consistent with this, combined SM09419 and venetoclax displayed synergistic anti-leukemic effects and venetoclax-sensitive AML cell lines (Fig.L). Taken together, these data support the notion of targeting CLK/DYRK1A in the context of BCL2 inhibition. In this study, we systematically defined gene interactions that mediate the response to a wide range of AML drugs. Recent studies have begun to show that dysfunctional RNA processing promotes AML development. However, the role of RNA processing in modulating drug responsiveness in AML is not well understood. Here, we have uncovered that synthetic lethal targeting of splicing factors, such as RBM10, increases sensitivity of AML cells to BCL2 inhibition. Therapeutically, pharmacologic inhibition of SR protein function via inhibiting CLK/DYRK1A-mediated phosphorylation of splicing factors is an effective strategy used in combination with venetoclax or to overcome venetoclax resistance. Overall, our findings underscore the central importance of RNA splicing in drug response and provides a therapeutic rationale for modulating RNA splicing to enhance current AML therapies. Figure 1 Figure 1. Disclosures McMillan: Prizer: Ended employment in the past 24 months. Bossard: Biosplice Therapeutics: Current Employment. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

OBJECTIVE: We discuss and evaluate surgical strategies and results in 42 patients with a variety of tumors involving the anterior and anterolateral foramen magnum and present factors affecting the degree of resection and patient outcomes. We describe our surgical techniques for resection of these tumors via the lateral approach, including consideration for occipital condylar resection and vertebral artery management. METHODS: A retrospective analysis was performed of 42 surgically treated patients with tumors involving the anterior and anterolateral foramen magnum. Patients received treatment between 1991 and 2002; patients’ files, operative notes, and pre- and postoperative imaging studies were used for the analysis. RESULTS: The female-to-male ratio was 28:14. Mean patient age was 47 years. Pathological entities comprised 18 meningiomas, 12 chordomas, 3 glomus tumors, 3 schwannomas, and 6 miscellaneous tumors. We mobilized the vertebral artery at the dural entry point in all patients with meningiomas. The vertebral artery was mobilized at the C1 transverse foramen for the majority of extradural tumors. Partial condyle resection was performed in eight meningiomas and five extradural tumors. Complete condyle resection was required in 12 cases, including 9 chordomas, 2 carcinomas, and 1 bone-invading pituitary adenoma. Thirteen patients required occipitocervical fusion after tumor resection. CONCLUSION: In anterior or anterolaterally located foramen magnum tumors, we think the extreme lateral or far lateral approach affords significant advantages. Vertebral artery mobilization and occipital condyle resection may be needed depending on the extent and location of the foramen magnum tumor and its specific pathological characteristics. Tumor invading the occipital condyle or significant condylar resection may cause occipitocervical instability and require fusion.