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Follicular lymphoma (FL) is a B-cell malignancy with a complex tumor microenvironment that is rich in nonmalignant immune cells. We applied single-cell RNA sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T cells, including a cytotoxic CD4 T-cell population. We characterized four major FL subtypes with differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced MHC expression on FL cells. In turn, expression of MHCII genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics of T cells. This provides a classification framework of the FL microenvironment in association with FL genotypes and MHC expression, and informs different potential immunotherapeutic strategies based upon tumor cell MHCII expression. Significance: We have characterized the FL-infiltrating T cells, identified cytotoxic CD4 T cells as an important component that is associated with tumor cell–intrinsic characteristics, and identified sets of targetable immune checkpoints on T cells that differed from FLs with normal versus low MHC expression. See related commentary by Melnick, p. 374. This article is highlighted in the In This Issue feature, p. 369

Supplementary Figure from Follicular Lymphoma Microenvironment Characteristics Associated with Tumor Cell Mutations and MHC Class II Expression

Supplementary Data from Follicular Lymphoma Microenvironment Characteristics Associated with Tumor Cell Mutations and MHC Class II Expression

Follicular lymphoma (FL) is a B-cell malignancy with a complex tumor microenvironment that is rich in nonmalignant immune cells. We applied single-cell RNA sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T cells, including a cytotoxic CD4 T-cell population. We characterized four major FL subtypes with differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced MHC expression on FL cells. In turn, expression of MHCII genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics of T cells. This provides a classification framework of the FL microenvironment in association with FL genotypes and MHC expression, and informs different potential immunotherapeutic strategies based upon tumor cell MHCII expression.Significance:We have characterized the FL-infiltrating T cells, identified cytotoxic CD4 T cells as an important component that is associated with tumor cell–intrinsic characteristics, and identified sets of targetable immune checkpoints on T cells that differed from FLs with normal versus low MHC expression.See related commentary by Melnick, p. 374.This article is highlighted in the In This Issue feature, p. 369

Supplementary figure legends and materials

Supplementary figure 1-2: Fig. S1. Fas mRNA in HCC cell lines and the effects of HuR knockdown on alternative Fas splicing. Fig S2. Effect of HuR knock-down on FasL-induced apoptosis in HepG2 cells.

Supplementary figure legends and materials

Purpose: To compare outcomes between patients with relapsed follicular lymphoma who received a nonmyeloablative allogeneic stem cell transplant (alloSCT) and those who received an autologous transplant (autoSCT). Patients and Methods: We evaluated 194 patients with follicular lymphoma who received an alloSCT (n = 98) or autoSCT (n = 96) at MD Anderson Cancer Center (Houston, TX). The transplant type used was based on donor availability and by Medicare reimbursement guidelines. Patients who received an alloSCT were enrolled in four consecutive trials in which they received fludarabine, cyclophosphamide (or bendamustine), and rituximab conditioning. autoSCT patients received R-BEAM (rituximab, carmustine, etoposide, cytarabine, and melphalan). Results: The median follow-up of survivors was 108 months for the alloSCT group and 102 months for the autoSCT group. Overall survival was significantly better for patients who received an alloSCT compared with those who received an autoSCT (62% vs. 46%; P = 0.048). Similarly, progression-free survival rates were 52% in patients who received an alloSCT and 31% in those who received an autoSCT (P < 0.001), and the 8-year relapse rates were 11% and 43%, respectively (P < 0.0001). Only three patients in the alloSCT group relapsed beyond 3.5 years. In the alloSCT group, the rates for grade 2 to 4 acute graft-versus-host disease (GVHD), grade 3 to 4 acute GVHD, and extensive chronic GVHD were 22%, 9%, and 38%, respectively. In the autoSCT group, the 8-year incidence of secondary myelodysplasia was 11%. Nonrelapse mortality was similar between the two groups (15% vs. 11% at 8 years; P = 0.27). Conclusions: This study shows that alloSCT is curative and confers superior survival compared with autoSCT in patients with follicular lymphoma.

Introduction: Prolonged grade 3-4 (G3-4) cytopenias lasting beyond day 30 have been observed with various chimeric antigen receptor (CAR) T-cell products. Their biologic mechanism remains largely unknown, since it is unclear whether it is related to the effects of the conditioning regimen or due to CAR T-cell activity. Methods: We measured 29 analytes associated with CAR-T cell activity by multiplex assays (V-Plex, Meso Scale Diagnostics) and CAR T-cell amplification by RT-PCR in serial plasma samples at baseline and days 7, 14, and 30 (D30) after standard-of-care axicabtagene ciloleucel (axi-cel) in patients with refractory large B-cell lymphoma (LBCL) treated at MD Anderson Cancer Center between March and August 2018. Patients with G3-4 cytopenia before conditioning, defined according to CTCAE v5, and those who progressed and/or died before D30 were excluded from the analysis. Unpaired t-test was used for area under the curve (AUC) and mean peak comparison, and the Benjamini-Hochberg method for adjustment of false discovery rate (FDR). Results: Of 19 patients included in the analysis, 9 (47%) had ongoing G3-4 cytopenia at D30. Baseline clinical characteristics were not significantly different between the 2 groups. Patients with D30 G3-4 cytopenia had a significantly higher AUC for the following cytokine levels: IFN-ɣ, FLT3-L, G-CSF, GM-CSF, CXCL1, IL-1Rα, IL-3, IL-8, IL-10, M-CSF, CCL2 (p Conclusions: Development of prolonged cytopenia after axi-cel in patients with LBCL is associated with significant increase in the levels of multiple inflammatory cytokines and with CAR T-cell amplification early after infusion, suggesting this is due to CAR T-cell activity rather than to the myelosuppressive effect of conditioning regimen. The majority of these cytokines appear to be derived from myeloid cells and peak at day 7, providing potential targets for prophylactic intervention in these patients. Citation Format: Paolo Strati, Nahum Puebla-Osorio, Guangchun Han, Jason R. Westin, Loretta J. Nastoupil, Sairah Ahmed, Felipe Samaniego, Nathan H. Fowler, Luis E. Fayad, Hun J. Lee, Christopher R. Flowers, Samer A. Srour, Grace Watson, Linghua Wang, Michael R. Green, Sattva S. Neelapu. Association between cytokine levels and prolonged cytopenia after axicabtagene ciloleucel in patients with refractory large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-57.

Introduction. Prolonged grade 3-4 (G3-4) cytopenias lasting beyond day 30 have been observed with various chimeric antigen receptor (CAR) T-cell products, suggesting a class effect. However, the mechanism(s) of prolonged cytopenias remains largely unknown, being unclear whether it is related to the effects of the conditioning regimen or due to CAR T-cell activity. Methods. To investigate whether CAR T-cell activity is associated with prolonged cytopenia, we measured 29 analytes associated with CAR-T cell activity by multiplex assays (V-Plex, Meso Scale Diagnostics) in serial plasma samples at baseline and days 7, 14 and 30 (D30) after standard of care axicabtagene ciloleucel (axi-cel) therapy in patients with refractory large B-cell lymphoma treated at MD Anderson Cancer Center between March and August 2018. Patients with G3-4 cytopenia before conditioning, defined according to CTCAE v5, and those who progressed and/or died before D30 were excluded from the analysis. Unpaired t-test was used for area under the curve (AUC) comparison, independent sample t-test for mean peak comparison, and the Benjamini-Hochberg method for adjustment of false discovery rate (FDR). Results. Of 19 patients included in the analysis, 9 (47%) had ongoing G3-4 cytopenia at D30. Baseline clinical characteristics were not significantly different between the 2 groups. Patients with D30 G3-4 cytopenia had a significantly higher AUC for the following cytokine levels: IFN-γ, FLT3-L, G-CSF, GM-CSF, CXCL1, IL-1Rα, IL-3, IL-8, IL-10, M-CSF, CCL2 (p≤0.05); IL-1β, IL-2Rα, IL-6, IL-15, TNF-α (p Conclusions. Development of prolonged cytopenia after axi-cel in patients with large B-cell lymphoma is associated with significant increase in the levels of multiple inflammatory cytokines early after CAR T infusion, suggesting this may be due to CAR T-cell activity rather than to the myelosuppressive effect of conditioning regimen. The majority of these cytokines appear to be derived from myeloid cells and peak at day 7, providing potential targets for prophylactic intervention in these patients. Analysis of CAR-T expansion is ongoing and will be presented at the meeting. Citation Format: Paolo Strati, Nahum Puebla-Osorio, Guangchun Han, Jason Westin, Loretta Nastoupil, Felipe Samaniego, Nathan Fowler, Luis Fayad, Hun Ju Lee, Christopher Flowers, Samer Srour, Grace Watson, Linghua Wang, Michael Green, Sattva Neelapu. Cytokines associated with prolonged Cytopenia after axicabtagene ciloleucel in patients with refractory large B-cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2049.

Background: ASTX660 is an oral, novel nonpeptidomimetic, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP). ASTX660 is currently being evaluated in a first-in-human phase 1–2 study in patients (pts) with advanced solid tumors or lymphoma (ClinicalTrials.gov NCT02503423). In the ongoing phase 2, ASTX660 has demonstrated preliminary evidence of clinical activity in the relapsed/refractory peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) cohorts (Mehta et al, presented at the EHA Conference 2019, abs PS1073). Here, we report overall efficacy and safety data from the solid tumors (head and neck squamous cell carcinoma [HNSCC]; cervical carcinoma and other solid tumors) and lymphoma (diffuse large B-cell lymphoma [DLBCL], PTCL, CTCL,) phase 2 cohorts. Methods: Pts received treatment with ASTX660 orally at the RP2D 180mg/day on days 1 to 7, and 15 to 22 in a 28-day cycle. In the first stage 14 evaluable pts were enrolled in each of the 6 phase 2 cohorts with the option to expand the cohort if activity was observed. The primary endpoint was response rate as assessed by the investigator according to the Lugano criteria (DLBCL and PTCL), Global Assessment (CTCL), or RECIST 1.1 (solid tumors). Adverse events (AEs) were assessed per CTCAE V4.03. Results: As of June 4, 2019, a total of 107 pts have received ASTX660 in the solid tumors and lymphoma phase 2 cohorts (HNSCC n=14; DLBCL n=16; PTCL n=26; CTCL n=23; cervical carcinoma n=14; other solid tumors n= 14). Median age (range) was 61 (23-84) years and median number (range) of prior anticancer regimens was 3 (0-12). Among all pts, the most common related AEs of any grade (≥ 10%) were rash (35%), lipase elevation (34%), amylase elevation (29%), diarrhea (14%), fatigue (14%), AST elevation (13%), nausea (13%), and anemia (11%). Related AEs ≥ Grade 3 occurring in ≥ 5% of pts were rash (18%), lipase elevation (16%) and amylase elevation (9%). As of 4 June 2019, 86 pts (80%) discontinued study treatment: 64 (60%) due to progressive disease, 13 (12%) due to AE, 4 (4%) due to death, 4 (4%) due to withdrawal by participant and 1 (1%) for investigator’s decision. At the time of analysis, the ORR was 36% in the PTCL cohort and 15% in the CTCL cohort. One PR was reported in a pt with metastatic melanoma after 12 cycles of treatment. No objective responses were reported in the HNSCC, DLBCL or cervical cohorts. Accrual in the PTCL and CTCL continues; updated efficacy and safety data will be presented at the meeting. Conclusion: In the phase 2 part of the study ASTX660 monotherapy has demonstrated a manageable safety profile and encouraging activity in PTCL and CTCL warranting cohort expansion. Future plans include evaluation of ASXT660 both as mono- or combination therapy in selected malignancies. Citation Format: Antoine Hollebecque, Sarit Assouline, Felipe Samaniego, Benoit You, Francine Foss, Anca Prica, Sarah W. Gordon, Marc Webster, Martin JS. Dyer, Dima El-Sharkawi, Geoffrey I. Shapiro, Lisa Nabell, Sarah P. Blagden, John Lister, Susanna V. Ulahannan, Yijun Sun, Danna Chan, Roberta Ferraldeschi, Monica Mita. Preliminary results of ASTX660, a novel non-peptidomimetic cIAP1/2 and XIAP antagonist, in 107 patients with solid tumors or lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A072. doi:10.1158/1535-7163.TARG-19-A072

Hepatocellular carcinomas (HCC) show resistance to chemotherapy and have blunt response to apoptotic stimuli. HCC cell lines express low levels of the Fas death receptor and are resistant to FasL stimulation, whereas immortalized hepatocytes are sensitive. The variable Fas transcript levels and consistently low Fas protein in HCC cells suggest posttranscriptional regulation of Fas expression. The 3′-untranslated region (UTR) of Fas mRNA was found to interact with the ribonucleoprotein Human Antigen R (HuR) to block mRNA translation. Silencing of HuR in HCC cells increased the levels of cell surface Fas and sensitized HCC cells to FasL. Two AU-rich domains within the 3′-UTR of Fas mRNA were identified as putative HuR-binding sites and were found to mediate the translational regulation in reporter assay. Hydrodynamic transfection of HuR plasmid into mice induced downregulation of Fas expression in livers and established functional resistance to the killing effects of Fas agonist. Human HCC tumor tissues showed significantly higher overall and cytoplasmic HuR staining compared with normal liver tissues, and the high HuR staining score correlated with worse survival of patients with early-stage HCC. Combined, the protumorigenic ribonucleoprotein HuR blocks the translation of Fas mRNA and effectively prevents Fas-mediated apoptosis in HCC, suggesting that targeting HuR would sensitize cells to apoptotic stimuli and reverse tumorigenic properties. Implications: Demonstrating how death receptor signaling pathways are altered during progression of HCC will enable the development of better methods to restore this potent apoptosis mechanism. Mol Cancer Res; 13(5); 809–18. ©2015 AACR.

Background: Rituximab (RTX) alone or in combination with chemotherapy has substantially improved treatment outcomes for patients (pts) with marginal zone lymphoma (MZL), but relapse is common and not all pts are acceptable candidates for, or respond to, current salvage therapies. Umbralisib is a novel, next-generation PI3K-delta inhibitor with unique inhibition of casein kinase-1ϵ (CK1ϵ) and, compared to earlier generation PI3K-delta inhibitors, exhibits a differentiated tolerability profile with reduced rates of immune-mediated toxicity (Burris et al, 2018). This registration-directed study evaluates the efficacy and safety of umbralisib in pts with relapsed/refractory (R/R) MZL. Methods: Pts had histologically confirmed MZL, ECOG PS ≤2, and had previously received ≥1 prior therapy including at least one CD20 monoclonal antibody (mAb)-containing regimen. All pts received umbralisib 800 mg orally once daily until progression or unacceptable toxicity. The primary study endpoint was overall response rate (ORR) as assessed by an independent review committee (IRC) according to 2007 IWG criteria. ORR by investigator assessment is reported here, and ORR by IRC is forthcoming. Secondary endpoints included duration of response (DOR), progression-free survival (PFS), and safety. Results: Sixty-nine pts were enrolled; we report on the first 38 who are eligible for at least 6 months (mos) of follow-up as of the data cutoff date. Disease status for the 38 pts: extranodal (n=23), nodal (n=8), and splenic (n=7). Median age was 67 years (range, 34-81). Median number of prior systemic therapies was 2 (range, 1-5). Seven pts (18%) had received monotherapy RTX only, and 26 (68%) had received at least one CD20 mAb-containing chemoimmunotherapy. As of the cut-off date, the median follow-up was 9.6 mos. Per investigator assessment, ORR was 55% (4 CRs and 17 PRs), 29% of pts (n=11) had stable disease (SD) of which 6 of these SD pts remain on study with durations ranging from 7-12+ mos. The clinical benefit rate (CR+PR+SD) was 84%, and 91% of pts with at least 1 post-baseline assessment experienced tumor reductions. The median time to initial response was 2.7 mos, while the median DOR was not reached (95% CI: 8.4-not reached). The 12-month PFS was 71%. The most common (≥20%) adverse events (AE) of any grade included: diarrhea (45%), nausea (29%), fatigue (26%), headache (26%), cough (24%), and decreased appetite (21%). The most common Grade 3/4 events were neutropenia (8%), febrile neutropenia (5%), and diarrhea (5%). As of the cutoff date, 16 pts discontinued treatment (PD: 18%; AEs: 8%; pt decision: 8%; physician decision: 8%) and 58% continue treatment. Conclusions: PI3K-delta inhibition with single-agent umbralisib is active and well tolerated in pts with R/R MZL, achieving durable responses with chemotherapy-free therapy. Citation Format: Nathan H. Fowler, Felipe Samaniego, Wojciech Jurczak, Ewa Lech-Maranda, Nilanjan Ghosh, Bertrand Anz, Piers Patten, James A. Reeves, Lori A. Leslie, Piotr Smolewski, Julio C. Chavez, Paolo Ghia, Corrado Tarella, John M. Burke, Jeff Sharman, Kathryn Kolibaba, Owen A. O'Connor, Chan Y. Cheah, Hari P. Miskin, Peter Sportelli, Michael S. Weiss, Pier Luigi Zinzani. Umbralisib monotherapy demonstrates efficacy and safety in patients with relapsed/refractory marginal zone lymphoma: A multicenter, open-label, registration directed Phase II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT132.

Purpose: The purpose of this study was to evaluate the clinical activity and toxicity of recombinant human Interleukin (IL)-12 in patients with relapsed and refractory non-Hodgkin’s lymphoma (NHL) or Hodgkin’s disease (HD). Experimental Design: Forty-two previously treated patients (32 patients with NHL and 10 patients with HD) were enrolled on the study. Patients were treated with either intravenous (n = 11) or subcutaneous (n = 31) administration of IL-12. The patients had received a median of three prior treatment regimens, and 16 patients had undergone prior autologous stem cell transplantation. Results: All patients were assessable for toxicity, and 39 of 42 (93%) patients were assessable for response. Six of 29 (21%) patients with NHL had a partial or complete response, whereas none of the 10 patients with HD responded. Furthermore, 15 patients had stable disease that lasted for up to 54 months. Progression-free survival in patients with indolent NHL, aggressive NHL, and HD was 6, 2, and 2.5 months, respectively. Treatment was well tolerated, and the most common toxicity was flu-like symptoms. Reversible grade 3 hepatic toxicity was observed in three patients requiring dose reduction. IL-12 therapy increased the median number of peripheral blood CD8 T lymphocytes from 423/μl to 576/μl (P = 0.0019). Furthermore, IL-12 therapy decreased serum vascular endothelial growth factor and basic fibroblast growth factor concentrations in 37% of the patients. Conclusions: The ability of recombinant human IL-12 therapy to increase the number of circulating CD8+ cells and induce clinical remissions in patients with relapsed NHL warrants further investigation of the drug.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of patients with DLBCL develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. The Bruton kinase (BTK) inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma (MCL) and DLBCL. Ibrutinib elicits overall desirable response rate with relapsed/refractory MCL and ABC DLBCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence and persistence of lymphoma is a problem. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines. In this study, IR DLBCL cell lines were generated by continuous culturing of parental (PT) cell lines in increasing concentrations of ibrutinib. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Of 5 ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC50>10µM).Three (TMD8, OCI-LY10, HBL1) were sensitive (IC50 < 10 nM). In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, showed elevated colony formation ability in methylcellulose matrix, and had a higher proliferation rate. Western blots and gene expression profile analysis showed increased expression by IR cell lines of inhibitors of apoptosis (IAP) family members, survivin, cIAP2, and oncogenic BCL2 and BCL6. The DNA damage repair pathway was found to be elevated with enhanced expression of CHK1 kinase. IR cell lines had reduced BTK expression without acquiring mutations, and downstream enhanced PI3K-Akt signaling. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated IR cell lines with the PI3Kβ/δ isoform-targeting drug KA2237 and observed reduced metabolic activity (survival) of PT cell lines and further reduction of surviving IR cell lines. In conclusion, this study highlights how changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL. Note: This abstract was not presented at the meeting. Citation Format: Neeraj Jain, Lalit Sehgal, Stephen Joseph Shuttleworth, Felipe Samaniego. Targeting PI3 pathway in ibrutinib resistant diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2366. doi:10.1158/1538-7445.AM2017-2366

Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Selective pressure provided by chemotherapy causes aberrant gene expression and selection of pre-existing population of chemo-resistant cells. These chemo-resistant cells can also transmit chemo-resistance to chemo-sensitive cells through exosomal transfer of molecules (miRNA, mRNA and proteins). In this study, we have analysed differentially expressed mRNA and miRNA from syngeneic ibrutinib (an irreversible BTK inhibitor, FDA approved) resistant DLBCL cells and from their isolated exosomes. Five DLBCL-GCB and five ABC subtype were analysed for their sensitivity with ibrutinib. We found three ABC subtype and one GCB subtype (OCL-LY1) were sensitive to ibrutinib. We have generated syngeneic ibrutinib drug resistant model from sensitive cells (at least 10 fold increase in IC50 value) by continuous culturing them in the presence of ibrutinib with gradual increase in drug concentration in each passage. Ibrutinib sensitive DLBCL cells when cultured with the syngeneic resistant cells in transwell apparatus, acquired resistance to ibrutinib. We also analysed the sensitivity of ibrutinib resistant cells with other conventional drugs for non-Hodgkin lymphomas. We further tested the expression of drug resistant genes in syngeneic resistant cells by real time PCR analysis. Later, we isolated the total RNA and miRNA species from DLBCL ibrutinib sensitive, their syngeneic resistant cells and from respective exosomes and analysed for differential expression by micro array analyses. Thus in conclusion, our study demonstrates that chemo-resistance can be transferred from resistant cells to sensitive cells via exosomal horizontal transmission of miRNA and mRNA in diffuse large B cell lymphoma cells. Moreover, our study identified potential targets that can be used to overcome chemo-resistance in diffuse large B cell lymphoma. Citation Format: Neeraj Jain, Lalit Sehgal, Tamer Khashab, Felipe Samaniego. Analysis of differentially expressed exosomal genes from ibrutinib resistant diffuse large B cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 306.

Mantle cell lymphoma is classified as distinct non-Hodgkin's lymphoma subtype characterized by the t(11;14)(q13;q32) translocation, which results in overexpression of Cyclin D1. The clinical presentation often includes extra-nodal involvement, of the bone marrow and gut. The prognosis of patients with mantle cell lymphoma (median overall survival, 3-5 years) is poorest among B-cell lymphoma patients. However, MCL pathogenesis has been delayed until the recent development of a tissue culture system, using stromal cells and adipocytes, suitable for propagating primary MCL cells. We hypothesized that tumor microenvironment might activate survival-signaling pathways essential for survival and maintenance of MCL, and targeting the identified signaling pathways might be an ideal for designing of curative treatment strategies. We developed an in-vitro culture system, mimicking bone marrow microenvironment that can be exploited to understand the biology of MCL resistance to death pathways. Primary MCL cells grown in co-culture with bone marrow stromal cells grew faster than cells cultured alone. We identified the soluble factors responsible for survival of MCL cancer stem cells (MCL-IC) that can be targeted to prevent relapse. We found the IL6 secretion triggered FGF-2 autocrine loop in MCL cells contributing to cell survival and growth by regulating mir101/EZH2/NFkb axis. The death signaling pathways inactive in MCL are restored upon therapeutic intervention by specific pathways inhibitors. In conclusion, we delineated three different signaling arm regulated in MCL cells to promote cell survival and resistance. We defined the factors supporting MCL and MCL-ICs survival essential for forming new targeting treatment strategies. To overcome MCL resistance to death pathways we have outlined the hMSCs-mediated support of MCL and identified drug targets to sensitize MCL to apoptosis. Citation Format: Lalit Sehgal, Neeraj Jain, Tamer Khashab, xin wang, Sattva Neelapu, Felipe Samaniego. Targeting mir101/ EZH2/NFkb axis by FGFR1 inhibitor in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3286.

Introduction Mantle cell lymphoma (MCL) represents an aggressive, incurable form of non-Hodgkin's lymphoma (NHL). The health complications associated with advanced age of MCL patients restrict treatment with intense chemotherapy. Translocation t(11;14), responsible for overexpression of cyclin-D1, is the hallmark of MCL. More detailed insight into MCL pathogenesis has been delayed until the recent development of a tissue culture system, using human mesenchymal stromal cells (hMSC), suitable for propagating primary MCL cells. We hypothesized that tumor-initiating cells are responsible for MCL relapse and chemoresistance and thus, identification of signals responsible for survival and maintenance of MCL-initiating cells (MCL-ICs) is essential for design of curative treatment strategies. Methods Isolates of primary MCL cells (n = 24) were co-cultured with human mesenchymal stem cells (hMSCs) and the content of MCL-ICs was analyzed by flow-cytometry based on marker expression profile; CD34-CD3-CD45+CD19-. Cytokine array was used to identify the soluble factors enriched in the co-cultures and the expression of these factors was confirmed by RT-PCR analysis. The signaling pathways employed by the newly-identified factors were blocked in 3 MCL cell lines (JVM2, Mino, Z138) to confirm their essential role in survival of MCL cells and, more importantly, for MCL-ICs. Results Co-cultures of primary MCL isolates with hMSCs supported the growth of MCL cells for over 4 weeks with continued presence of MCL-ICs (CD34-CD3-CD45+CD19-) representing about 1% of MCL cells. We found that IL-6 produced by hMSCs triggered an FGF/FGFR autocrine loop in MCL-ICs. The extent of FGFR expression correlated tightly with expression of SOX11, a pathology related negative prognostic marker in MCL. MCL cell survival and growth was regulated via the FGFR/mir101/ EZH2/ NF-κB/XIAP axis. Blocking of this signaling pathway with FGFR1 inhibitors consistently induced early reduction in XIAP levels and subsequently MCL cell death. Conclusion We established that propagation of primary MCL in co-cultures with hMSCs depends on an FGF/FGFR autocrine loop that enhances XIAP protein expression and thus, supports survival of MCL cells. We identified the factors essential for survival of MCL and MCL-ICs that present new targets for improved MCL treatment strategies. Citation Format: Lalit Sehgal, Rohit Mathur, Zuzana Berkova, Tamer Khashab, Xin Wang, Jorge E. Romaguera, Alma E. Rodriguez, Sattva Neelapu, Felipe Samaniego. Tumor microenvironment influences survival of mantle cell lymphoma-initiating cells through FGF/FGFR1 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2015-445

Objective: Many genotoxic therapies, including radiation, depend on intact Fas signaling to eradicate cancer cells. Defective Fas signaling is an important cause of cancer resistance to therapy. Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectrometry was used to identify Fas-associated proteins; co-immunoprecipitation and Western blot were used to detect interactions of PMLRARα, PML, c-FLIP and Fas, and to examine the components of death-inducing signaling complex (DISC) and caspase-8 cleavage. Deletional mutagenesis was used to map the interaction domains. PML shRNA lentivirus and As2O3 were used to knock down PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis. Mice were transfected with PMLRARα, monitored for survival, and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase-8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4965. doi:1538-7445.AM2012-4965

Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia-retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms a complex with Fas associated with suppression of Fas signaling. These data suggest that cancer-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2908.

The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas is not known. K1 is known to activate Akt and nuclear factor kappa B (NF-kB) through immunoreceptor tyrosine-based activation motif (ITAM) and may also bind to Fas receptor through its immunoglobulin (Ig) chain-like domain and interfere with apoptosis. We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling. Examination of mice expressing K1 via a ubiquitous promoter showed that 90% K1 transgenic mice (n=10) had developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. In vitro overexpression of an Ig domain-containing protein CD79b or treatment cells with K1 peptides revealed competition with K1-Fas binding in a dose-dependent manner and rate enhancement of Fas-mediated apoptosis. We have also shown that K1 suppressed Fas-mediated apoptosis, even in cells that did not express K1. Transfection of K1 into one pool of mouse cells protected against Fas-mediated apoptosis of a second pool of human Fas-transfected mouse cells indicating protection in trans. This analysis indicates a key role of K1 in suppression of Fas-mediated apoptosis which operates in a cis and trans protective role against apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1254.