Manske, Michelle K.

AbstractErdheim–Chester disease (ECD) is a histiocytic neoplasm that predominantly harbors mitogen‐activated protein kinase (MAPK) pathway variants. MAPK inhibitors typically are effective treatments, but mutations outside the MAPK pathway, such as CSF1R variants, may cause refractory ECD. We describe a patient with a novel somatic mutation in CSF1R (CSF1RR549_E554delinsQ) that resulted in refractory ECD affecting the central nervous system. Cell model studies, RNA sequencing analysis, and in silico protein modeling suggested that she had a gain‐of‐function mutation occurring in a region critical for autoinhibition. The patient was treated with pexidartinib, a CSF1R inhibitor, and has had a complete clinical and metabolic response lasting more than 1.5 years to date. To our knowledge, this is the first report to describe successful treatment of a patient with ECD by using an agent that specifically targets CSF1R. This case also highlights the critical role of individualized molecular profiling to identify novel therapeutic targets in ECD.

Abstract Introduction: DNA mismatch repair (MMR) is a major mechanism cells use to repair erroneous insertion or deletion of bases during DNA replication. MMR deficiency (MMR-d), including rare germline mutations (Lynch syndrome) and somatic inactivation, has been extensively studied in solid tumors, such as colon, endometrial, pancreatic, and ovarian cancers. The prevalence of MMR-d in solid tumors ranges from 1% - 20% of somatic defects, and patients with MMR-d tumors have a longer overall survival. It is also well-known that MMR is an integral part of activation-induced cytidine deaminase (AID)-mediated somatic hypermutation and immunoglobulin class switch processes in normal mature B lymphocytes, while mice deficient in the MMR genes are prone to develop T and B cell lymphomas. It is also known that follicular lymphoma (FL) carries a high degree of somatic hypermutation with some heterogeneity induced by AID. Our understanding of MMR status in human blood cancers, especially in FL remains very limited. One could hypothesize that MMR-d status could permit cells to acquire and sustain high-degree of mutation rates, better sensitivity to DNA damaging chemotherapy while hindering rapid cell proliferation, which could manifest to a better prognosis in patients. Herein, we conducted an exploratory analysis to understand the frequency of MMR-d status and its prognostic significance in FL. Methods: Following approval of the Institution Review Board, we assessed the expression of MMR proteins; MLH1, PMS2 and MSH6 on formalin fixed paraffin embedded (FFPE) lymph node tissue obtained from patients with FL by immunohistochemistry (IHC) using standard methods on Ventana Benchmark XT automated stainers (Ventana Medical Systems, Tucson, AZ). Dako (clone ES05), Cell Marque (clone EPR3947) and Biocare (clone BC/44) antibodies were used to stain MLH1, PMS2 and MSH6, respectively. Fifty cases of FL with existing FFPE blocks were selected randomly. The expression of MLH1, PMS2 and MSH6 proteins in the tumor cells was scored as 0, +1 or +2, (Figure 1A). Cases with absence of the IHC signal (score of 0) in any one of the three proteins were classified as MMR-d. Progression free survival at 12 months (PFS12) or 24 months (PFS24) was defined as being free from disease progression or death at 12 or 24 months, respectively. All time-to-event analyses were done from the time of diagnosis. Results: Fifty patients with FL were included in the study. The median age at diagnosis was 65 years (range 34-85) and 58% were females. At initial diagnosis, 36 (72%) patients had advanced stage (stage 3, n=8, stage 4, n=28) disease. Of the 50 patients, 26 (52%) required treatment; chemotherapy (n=14), radiation treatment alone (n=9), surgery alone (n=3). Out of the 50 patients, 23 (46%) were classified as MMR-d due to lack of expression of one of the three assessed proteins: MLH1 (n=1, 2%), PMS2 (n=11, 22%) and MSH6 (n=19, 38%). The patient who had MLH1 loss had concomitant PMS2 loss. Stage IV disease was observed in 52% of the MMR-d patients and 59% in the MMR proficient (MMR-p) patients, p=0.77. Median age at diagnosis was 62 (range: 34-84) years and 67 (range: 34-85) years in MMR-d and MMR-p patients, respectively, p=0.1. FLIPI score was similar between the MMR-d [low n=10 (43%), intermediate n=9 (39%), high n=4 (18%)] and MMR-p [low n=5 (18%), intermediate n=14 (52%), high n=8 (30%)] patients, p=0.16. Fourteen (61%) of MMR-d patients required initiation of therapy upon diagnosis compared to 12 (44%) of MMR-p patients, p=0.27. The median follow-up for the entire cohort was 17 years [95% confidence interval (CI): 11.7-29.4]. The median overall survival (OS) of MMR-d patients was 10 years (95%CI: 7.8-20.7) compared to 6 years (95%CI: 4.5-NR) in MMR-p patients, p=0.036, Figure 1B. In patients with MMR-d and MMR-p, 83% and 55% achieved PFS12, p=0.04 and 65% and 41% achieved PFS24, p=0.08, respectively. After adjusting for FLIPI score, MMR status remained associated with OS (MMR-d HR: 0.51, 95%CI: 0.2-1.02, p=0.05). Discussion and Conclusion: In this pilot study, MMR-d in the FL tumors was common (46%) and, as predicted, was associated with a favorable OS. Association of MMR-d status with PFS in general and patients with early progression (short PFS12 and PFS24) and relationship of MMR-d with types of therapy need further study in large FL datasets. Figure 1 Figure 1. Disclosures Paludo: Karyopharm: Research Funding. King: Celgene/BMS: Research Funding. Maurer: Celgene: Research Funding; Genentech: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Nanostring: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nowakowski: Celgene, NanoString Technologies, MorphoSys: Research Funding; Celgene, MorphoSys, Genentech, Selvita, Debiopharm Group, Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Witzig: Karyopharm Therapeutics, Celgene/BMS, Incyte, Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS, Acerta Pharma, Kura Oncology, Acrotech Biopharma, Karyopharm Therapeutics: Research Funding.

Abstract Introduction: Low-grade B-cell lymphomas (LGBCL), aside from follicular lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma, account for approximately 10% of B-cell non-Hodgkin lymphomas and consist of several subtypes. While a majority of LGBCL cases have an overall favorable prognosis, we have previously shown that cases who have an event (relapse or progression, transformation, or re-treatment) within 24 months of diagnosis (EFS24) have an inferior overall survival (OS) compared to those achieving EFS24 (Tracy et al., AJH 2019;94:658-66). However, the underlying biological characteristics associated with early failure and aggressive disease across LGBCL subtypes are unknown. In this study, we used matched transcriptomic, genomic, and immune profiling data from LGBCL cases, the largest cohort to date, and asked whether there were unique biological phenotypes across different LGBCL subtypes and whether we could identify signatures associated with aggressive LGBCL. Validation of the prognostic utility of this signature was performed on a previously published, independent cohort of 63 pre-treatment LGBCL cases. Methods: Tumors from 64 newly diagnosed LGBCL patients from the Molecular Epidemiology Resource of the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence were included in this study (SMZL (n = 48), NMZL (n = 6), LPL (n = 5), B-NOS (n = 3), EMZL (n = 2)). RNA sequencing (RNAseq) data from 61 LGBCL tumors and 5 benign CD19+CD27+ memory B samples was subjected to NMF clustering to define groups. Differential expression and pathway analysis were used to identify biological characteristics of each cluster. CIBERSORT was used to identify immune cells in the tumor microenvironment. Whole exome sequencing (WES) was performed on 61 tumor-normal pairs. Singscore was used to assign a single score per patient representing gene expression of the survival-associated transcriptomic signature identified in this study. Results: NMF analysis of RNAseq data identified 5 clusters of patients, denoted LGBCL1-5 (Fig 1A). Patients from the same diagnostic subtype did not exclusively cluster together, with all LGBCL clusters comprised of patients from multiple subtypes (Fig 1B). Exploring the association between patient cluster and outcome, we observed significantly inferior event-free survival (EFS) (HR 2.24; 95% CI 1.01-4.98) and overall survival (OS) (HR 5.59; 95% CI 2.00-15.63) in LGBCL5 patients compared to LGBCL1-4 (Fig 1C). In addition, 80% of the transformation cases in our cohort were classified as LGBCL5 (Fig 1D). Differential expression and pathway analysis showed distinct processes significantly upregulated in each cluster (FDR < 0.05), with LGBCL5 demonstrating enrichment of cell cycle and mitosis pathways. CIBERSORT identified increased immune cell content in LGBCL3 and LGBCL5 compared to other clusters, with high frequencies of mast cells in both (p = 0.0002), increased CD8 T cells in LGBCL3 (p < 0.0001), and increased T follicular helper cells in LGBCL5 (p = 0.004). WES identified previously reported alterations in NOTCH, NFkB, and chromatin remodeling pathways and novel variants in LGBCL, including mutations in HNRNPK, CLTC, HLA-A, HLA-B and HLA-C. Assessment of alterations by cluster showed significant enrichment of TNFAIP3 (OR 5.54; 95% CI 1.20-28.14) and BCL2 alterations (OR 5.49; 95% CI 1.07-32.02) in LGBCL5 cluster. Finally, we identified a cell cycle-related transcriptomic signature of 108 genes upregulated in LGBCL5 and EFS24 failure cases. Cases with high expression of this signature showed significantly inferior EFS (HR 14.25; 95% CI 4.90-41.38) and OS (HR 7.82; 95% CI 2.40-25.44) compared to cases with low expression in our discovery cohort. This observation was reproduced in an independent validation cohort, where patients with high expression of this signature demonstrated significantly inferior EFS (HR 5.70; 95% CI 1.49-21.79) and OS (HR 10.07; 95% CI 2.00-50.61). Conclusions: In this study, we are the first to define mechanisms of pathogenesis in LGBCL with shared transcriptomic, genomic, and immune profiles present across LGBCL subtypes. We then further defined a gene expression signature associated with inferior patient outcome, with application of this signature to an independent validation cohort demonstrating proof of concept and utility of this signature as a prognostic marker in LGBCL patients. Figure 1 Figure 1. Disclosures Maurer: Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Nanostring: Research Funding. Paludo: Karyopharm: Research Funding. Habermann: Tess Therapeutics: Other: Data Monitoring Committee; Morphosys: Other: Scientific Advisory Board; Incyte: Other: Scientific Advisory Board; Seagen: Other: Data Monitoring Committee; Loxo Oncology: Other: Scientific Advisory Board; Eli Lilly & Co.,: Other: Scientific Advisor. Link: MEI: Consultancy; Genentech/Roche: Consultancy, Research Funding; Novartis, Jannsen: Research Funding. Rimsza: NanoString Technologies: Other: Fee-for-service contract. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Cerhan: Genentech: Research Funding; Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding. Novak: Celgene/BMS: Research Funding.

Abstract Background: Follicular lymphoma (FL) exhibits significant clinical, cellular, molecular, and genetic heterogeneity. Our understanding of FL biology and molecular classifications of FL, to date, has largely been driven by pathologic classification (Grade 1-3b), genetic classification (m7-FLIPI), or gene expression profiling (IR-1/2; Huet-23), along with limited studies on small cohorts or targeted panels. In order to further understand the biological underpinnings and complexity of FL, large-scale and integrated whole exome sequencing (WES) and RNA sequencing (RNAseq) studies are needed. Using a highly-annotated cohort of 93 FL tumors with matched RNAseq, WES, and CyTOF data, we have explored the transcriptomic signature of purified FL B cells and identified unique molecular subsets that are defined by distinct pathway activation, immune content, and genomic signatures. Methods: Frozen cell suspensions from 93 untreated FL (Grade 1-3b) patients' tumor biopsies, enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE, were used for the study. DNA was isolated from whole tumor cell suspensions, and RNA was isolated from both whole tumor and B cell-enriched cell suspensions. RNAseq and WES were performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using a standard pipeline and novel driver genes were identified using Chasm+ driver analysis. Copy number variants were identified from WES data using GISTIC 2.0. NMF clustering and single sample gene set testing for B cell lineage and tumor microenvironment (TME) signatures were performed in R using the NMF and singscore packages. Results: Unsupervised clustering of RNAseq data identified three distinct expression programs which separated patient B cell samples into 3 groups: Group 1 (G1, n=20), Group 2 (G2, n=24), Group 3 (G3, n=43). While no clinical attributes were defined by any single group, G1 consisted of cases that had less aggressive characteristics (63% Stage I-II, 79% FLIPI 0-1). To identify unique transcriptional pathways driving the three expression programs, we scored gene level contributions to NMF expression programs and employed gene set enrichment analysis. This revealed significant pathway association with type-I IFN signaling (G1), DNA repair and stress response (G2), and epigenetic modulation (G3) as differentiating programs between the 3 groups (FDR q<0.001). VIPER master regulator activity inferencing revealed that these pathways were likely being controlled by differential activity in NF-kB, IRFs, STAT1, BCL6, and FOXO1. Each program significantly enriched for, but were not defined by, portions of specific germinal center programs, such as pre-memory (G1), light-zone-to-dark-zone transition (G2), and a pre-light-zone intermediate (G3). We next assessed the connection between B cell programs and the tumor microenvironment (TME) using available paired CyTOF data on 67 cases, which revealed an active TME in G1, with an abundance of CD8 T cell and NK cell populations, a wide variety of immune content in G2 that consisted mostly of Tfh and myeloid cells, and a poorly populated immune compartment in G3 compared to G1 and G2. Finally, somatic driver mutations and copy number alterations from WES were identified and associated with the three clusters. The three groups distinguished themselves by significant enrichment of copy number alterations (TNFAIP3-loss , 1q23-gain, 1q32-gain) in G2, while 10q-loss and mutations in BCL2 and chromatin modifiers (KMT2D and CREBBP) enriched in G3. G1, overall, had lower alteration burden and had weak associations with any specific alterations, suggesting an alternative mechanism for driving the G1 program. Conclusion: In this study, we have identified three unique FL tumor B cell groups, defined by specific transcriptional programs. Pathways such as inflammation, DNA damage response, and chromatin modification contribute most to the differences between B cell samples and group membership. Additionally, each program associated with specific genetic events as well as TME composition, highlighting potential drivers of these B cell states. This study improves the understanding of the mechanisms driving FL tumors and motivates further investigation into transcriptional consequences of genetic events as well as potential tumor intrinsic factors that may influence the TME. Figure 1 Figure 1. Disclosures Maurer: BMS: Research Funding; Genentech: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding. Rimsza: NanoString Technologies: Other: Fee-for-service contract. Link: MEI: Consultancy; Genentech/Roche: Consultancy, Research Funding; Novartis, Jannsen: Research Funding. Habermann: Tess Therapeutics: Other: Data Monitoring Committee; Seagen: Other: Data Monitoring Committee; Incyte: Other: Scientific Advisory Board; Morphosys: Other: Scientific Advisory Board; Loxo Oncology: Other: Scientific Advisory Board; Eli Lilly & Co.,: Other: Scientific Advisor. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. King: Celgene/BMS: Research Funding. Cerhan: Genentech: Research Funding; Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding. Novak: Celgene/BMS: Research Funding.

AbstractBackgroundDespite the unprecedented success of ibrutinib in lymphoma therapy, the development of ibrutinib resistance due to acquired BTK or PLCγ2 mutations has become a new clinical problem. However, not all resistance is mediated by these mutations and these mechanisms are poorly understood due to a lack of study tools that truly recapitulate this clinical scenario.MethodsWe established a novel patient‐derived ibrutinib‐resistant mantle cell lymphoma (MCL) line named MCIR1. Using immunological, molecular, and cytogenetic approaches, we comprehensively characterized MCIR1 and further demonstrated its utility in the study of resistance mechanisms and treatments to overcome this resistance.ResultsWe show that MCIR1 is a bona fide ibrutinib‐resistant MCL cell line with normal BTK‐/PLCγ2 but ibrutinib‐resistant ERK1/2 and AKT1 signaling. RNA‐Seq analysis revealed a robust non‐canonical NF‐kB signaling that drives the ibrutinib resistance. We also demonstrate the potential utility of a MCIR1‐based cell and mouse model for the discovery of new treatments to overcome BTK inhibitor resistance.ConclusionsWe have established the first patient‐derived ibrutinib‐resistant MCL cell line MCIR1 that lacks BTK or PLCγ2 mutations but exhibits a hyperactive non‐canonical NF‐kB pathway. We further demonstrate its utility in the discovery and validation of new drugs to overcome this resistance.

Background: Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and presents with significant clinical, cellular, molecular, and genetic heterogeneity. Despite the name and defining microanatomic location, the genetic and molecular identity and pathogenesis of the FL tumor cell is largely uncharacterized. Prior clinical and molecular classifications of FL have been primarily driven by pathologic classification (Grade 1-3b), genetic classification (M7-FLIPI), or gene expression profiling (IR-1/2). Using a unique cohort of 93 FL tumors, we have explored the transcriptomic signature of purified FL B cells, along with their matched whole tumor, and identified unique molecular subsets which are defined by distinct pathway activation, immune content, and genomic signatures identified through whole exome sequencing (WES). Methods: Frozen tumor biopsies from 93 untreated FL (Grade 1-3b) patients enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE were used for the study. DNA was isolated from whole tumor cell suspensions and RNA was isolated from both whole tumor and B cell enriched cell suspensions. RNA sequencing (RNAseq) and WES was performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using the Mayo Clinic standard pipeline and novel driver genes were identified using 20/20+ driver analysis. Copy number variants were identified using GISTIC 2.0. NMF clustering and single sample gene set testing, for B cell lineage and tumor microenvironment (TME) signatures, was performed in R using the NMF and SingScore packages. Results: NMF consensus clustering of FL B cell RNAseq data identified two distinct subsets, C1 (n=32) and C2 (n=57). Clinically, C1 was associated with being FL grade 3 (p<0.001) and more aggressive therapy (e.g. immunochemotherapy) at diagnosis while other clinical factors were similar between the groups. To determine the biologic underpinnings that drove the NMF analysis, the top NMF metagenes were incorporated (n=389 for C1 and n=293 for C2) for GO analysis. Among those genes were AICDA, IRF4, and EZH2 in C1, and BCL2 in C2. Additionally, we identified significant gene set enrichment with NMF metagenes and calculated differential gene set expression comparing C1, C2, and normal B cells (n=5). Together, these analyses suggest C1 displays significantly (p<0.05) enhanced DNA replication and metabolic activity, whereas C2 displays significantly enhanced cytoskeleton rearrangement, repressed inflammatory cytokine signaling, and NF-kB activity. Based on recent scRNAseq data suggesting that FL derives from distinct germinal center B cells, the B lineage difference between C1 and C2 was explored. Both subgroups were heterogeneous for dark (DZ), intermediate (INT), light (LZ) zones, and plasmablast (PBL) gene signatures. When compared to benign B cell samples, C1 was enriched for a pre-PBL (p<0.01), and C2 for an INT phenotype related to a LZ signature (p<0.01). To define if a genomic signature was associated with C1 and C2, mutations and copy number variants (>5% frequency) were examined. TNFAIP3, TP53, and BCL6 alterations were enriched in C1 samples, whereas C2 associated with alterations in BCL2, KMT2D, CREBBP, REL, and MYC. Finally, B cell clusters were analyzed for TME signatures. C1 samples displayed significant enrichment of macrophage, cytotoxic cell, gamma-delta-Tcell, and endothelial cell TME elements (p<0.05) consistent with an inflamed/hot tumor. In contrast, C2 samples exhibited significantly lower signatures (p<0.05) in all entities except for the fibroblast signature, compared to C1 and normal, consistent with an immune desert phenotype. Conclusion: Our results suggest that B cells from FL patients display two distinct transcriptomic signatures. C1 identifies an immunologically active tumor, driven by TNFAIP3 alterations, with pre-PBL characteristics, DNA replication and repair, inflammatory cytokine secretion/signaling, and hyper-metabolic characteristics. C2 identifies an immunologically quiet tumor, driven by alterations in BCL2 and chromatin modifiers, with an intermediate GC phenotype, repressed cytokine signaling, and active cell cycle progression and cytoskeleton rearrangement. This study improves our understanding of the mechanisms driving FL tumors and motivates further investigation into the relationship between tumor intrinsic factors that may influence the TME. Disclosures Maurer: Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding; Celgene / BMS: Research Funding. Ansell:ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; AI Therapeutics: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Novak:Celgene/BMS: Research Funding.

Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (selinexor), a first in class CRM1 inhibitor recently approved for multiple myeloma and diffuse large B-cell lymphoma (DLBCL), has been limited by substantial adverse effects (AEs). To address this clinical problem, we focused on identifying novel strategies to boost the potency, reduce toxicity, and broaden the applicability of CRM1 inhibitors to a wider range of malignancies. We discovered that salicylates could markedly enhance the anti-tumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. KPT-330 was chosen as the prototypical CRM1 inhibitor given its current FDA approval status and characterized pharmacokinetics; and choline salicylate (CS) was chosen as the prototypical salicylate given its favorable pharmacokinetics and reduced antiplatelet, renal, neurological and gastrointestinal AEs in humans compared to other salicylates. By using cell lines belonging to different hematologic malignancies and solid tumors, we demonstrated ex vivo that the combination of KPT-330 and CS (K+CS) could induce unique and significant antitumor effect at much lower dose of KPT-330 (at 25% of the dose used in the clinic), thereby potentially mitigating prohibitive clinical AEs (Figure 1a-d, and e-g). This significant synergetic antitumor effect observed with K+CS ex vivo was also validated in vivo by using an NSG mouse model of mantle cell lymphoma (Figure 1h). Moreover, the K+CS combination did not show this potent toxic effect on non-malignant cells in vivo and was safe without inducing toxicity to normal organs in NSG mice. Mechanistically, protein profiling through mass spectroscopy revealed that K+CS uniquely affects the cellular pathways of DNA damage repair, DNA synthesis and cell cycle progression. Studies involving immunoblotting, cell cycle analysis, immunofluorescence microscopy assessing nucleocytoplasmic molecular export and DNA damage, reporter assay to assess homologous recombination repair proficiency and immunohistochemistry showed that, compared to KPT-330 treatment alone, K+CS decreased the expression of CRM1, Rad51 and thymidylate synthase proteins in vitro and in vivo, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, and importantly a unique cell cycle arrest in S-phase, thus leading to cell apoptosis. These effects on cellular proteins and pathways were unique to K+CS treatment and were not observed with KPT-330 or CS single agent treatment. Pathway analyses through RNA sequencing also paralleled the findings of proteomic studies thus further validating the unique effect of K+CS treatment on the aforementioned cellular pathways. Importantly, K+CS treatment exerted unique and significant antitumor effect ex vivo on primary malignant cells obtained from patient's with high-risk hematologic malignancies such as double hit DLBCL, BTK and BCL2 inhibitor resistant mantle cell lymphoma, TP53 deleted/mutated chronic lymphocytic leukemia and high-risk multiple myeloma thus signifying its broader applicability as a treatment option for these aggressive hematologic malignancies where there is a dire need to find new treatment strategies (Figure 1i). In summary, we describe a unique all-oral drug combination with a novel constellation of mechanisms of action on cellular pathways that are exploited by cancer cells. K+CS represents a new class of therapy for multiple cancer types and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for therapy of blood cancers. Disclosures Witzig: Karyopharm Therapeutics: Research Funding; Acerta: Research Funding; Incyte: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Research Funding; MorphSys: Consultancy; Immune Design: Research Funding; Spectrum: Consultancy.

Abstract Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

Abstract MALT lymphoma is a genetically unique disorder and five mutually exclusive chromosomal translocations have been identified thus far in this disease: t(11;18), t(14;18), t(1;14), t(1;2) and t(3;14). Despite this genetic heterogeneity, all but one of the translocations affect the NF-κB signaling pathway, which is critical if not essential for antigen receptor mediated B- and T-cell activation and likely enhances MALT lymphoma growth. However, the known translocations are present in only 25% of cases suggesting that additional uncharacterized translocations exist. We used long-distance inverse polymerase chain reaction (LDI-PCR) technique to clone a novel IGH translocation partner in a 60-year-old female with a history of Sjogren’s syndrome and primary MALT lymphoma involving the parotid gland. The breakpoint on chromosome 14 occurred within the IGHSA2 switch region while the breakpoint on chromosome X fell within Xp11.4. The breakpoint on chromosome Xp11.2 fell between two genes, GPR82 and GPR34, both of which code for orphan G-protein coupled receptors (GPCRs). The breakpoint also fell within and disrupted a larger gene called CASK (a membrane-associated guanylate kinase-MAGUK). To determine the prevalence of this translocation in MALT lymphoma we performed interphase FISH studies on 64 MALT lymphomas using a breakapart probe for GPR82. Only the index case had an abnormal split signal pattern. We next designed primers to perform real-time quantitative RT-PCR for the genes located on Xp11 and found that GPR34 RNA was highly expressed, a 49-fold increase, in the patient carrying the t(X;14)(p11.4;q32) translocation compared to a normal splenic B cell control. Expression of GPR82 and CASK RNA was similar between normal B cells and the patient carrying the t(X;14)(p11.4;q32) translocation. These data suggest that the GPR34 gene is dysregulated upon its translocation to the IGHSA2 switch region. We then measured GPR34 RNA expression in a panel of MALT lymphomas (n=12) and found that GPR34 was expressed at levels higher than that seen in normal B cells with an average increase of 11 fold, 9/11 of which had an expression lever greater than 3-fold over normal splenic B cells. In a gastric MALT lymphoma specimen arising in a 67-year-old male, we saw a 64 fold increase in GPR34 expression. Interphase FISH studies performed on this specimen showed an extra intact GPR34 signal but no translocation involving IGH or GPR34, suggesting that other mechanisms, including gene dosage effect, can upregulate GPR34. GPR34 RNA was also detected in other normal B cell populations and histologic subtypes of NHL but not to the extent seen in MALT lymphoma; values represent the expression of GPR34 normalized to β-actin and a normal B cell control (value of 1.0): t(X;14)(p11.4;q32) specimen, 49.0; MALT lymphoma, 11.0 (n=12); peripheral blood B cells, 0.48 (n=2); normal bone marrow B cells, 0.97 (n=3); follicular lymphoma, 2.47 (n=3); marginal zone B cell lymphoma, 2.47 (n=3); diffuse large cell lymphoma, 0.36, (n=3); mantle cell lymphoma, 2.67 (n=3); and multiple myeloma 0.52 (n=6). The receptor encoded by GPR34 is most similar to the PY2 receptor subfamily of GPCR and GPR34 mRNA transcripts are particularly abundant in mast cells while lower levels were detected in other immune cells including B cells. However, little is known about its natural ligand, biologic function, or the signaling cascades activated by its engagement. Because the NF-κB signaling pathway has been shown to be a common downstream target of MALT lymphoma translocations we first examined the impact of GPR34 expression on phosphorylation of Iκ-Bα. Transient expression of a YFP-GPR34 expression plasmid in HeLa cells results in increased phosphorylation of Iκ-Bα compared to the YFP control. Additionally, we observed increased phosphorylation of ERK1/2 in GPR34-expressing cells, however no change in phosphorylation of GSK3β was detected. In summary, these data identify a novel IGHS translocation partner in MALT lymphoma and suggest that dysregulation of GPR34 is commonly found in MALT lymphoma. Overexpression of GPR34 results in activation of the NF-κB and MAP kinase pathways and may be a novel mechanism by which MALT lymphoma occurs.