Wu, Xiaosheng

TNF receptor-associated factor 3 (TRAF3) is an adapter protein that inhibits many signals that promote B cell survival and activation. Mice with a B cell-specific TRAF3 deficiency and humans with a rare haploinsufficiency in TRAF3 have enhanced development of BCLs as they age. Loss-of-function mutations in TRAF3 are common in B cell malignancies. Recent studies show that pharmacological inhibition of the enzyme glycogen synthase kinase 3 (GSK3), which regulates cellular growth, survival, and metabolism, inhibits growth and survival of BCL-derived B cells. In this study, we found that TRAF3 and GSK3 associated in B cells. The relative levels of TRAF3 in BCL cell lines correlated positively with the ratio of inactive to total GSK3β, and negatively correlated with susceptibility to GSK3 inhibition by the GSK3 inhibitory drug 9-ING-41, currently in clinical trials. Uniquely in BCLs with low TRAF3, GSK3 inhibition caused increased loss of the TRAF3-regulated, anti-apoptotic protein Mcl-1. GSK3 inhibition also blocked hyperresponsiveness to IL-6 receptor signaling in TRAF3-deficient BCL cells. Together, these results support the utility of 9-ING-41 as a treatment for BCL, and suggest that a decrease or loss of TRAF3 in BCLs could act as a biomarker for increased susceptibility to GSK3 inhibitor treatment.

Abstract Purpose: Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied. Experimental Design: Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays. Results: We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition. Conclusions: IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease.

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 Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

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.

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.

SPOP mutation disables 53BP1 ubiquitination and exclusion from DNA double-strand break sites and causes genome instability.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p < 0.001) [Fig 1A]. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p<0.01, Fig 1C). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and CHK2 (T68) (data not shown). This finding was further confirmed by WB analyses (Fig1DI). In addition, decreased of total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY19 and OCI-LY17 cell lines (Fig 1DI). Immunoprecipitation experiments with anti-PD1 antibody showed that PD-1 was in the same complex with SHP-1 and SHP-2 in OCI-LY7 cells but only with SHP-2 in OCI-LY19 cells (Fig 1DII). In parallel, mRNA sequencing was performed on 11 nodal tissues from either RT (n=8) or progressive CLL (n=3) after they developed clinical progression. The expression of PD-1 (PDCD1) was positively correlated with the expression of SHP-1 (PTPN6, r=0.56) and Cyclin E1 (CCNE1, r=0.62), implicating a direct role of PD-1 in regulating cellular proliferation and the induction in phosphatase expression in RT and CLL. Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.