ALBERT

Description: Background: Numerous studies have shown that lymphoma B-cells are resistant to CTL-mediated death, however the underlying mechanism for this resistance is not clear. In previous work, we have identified a subset of CD4+CD25+ T-cells overrepresented in the tumor sites of B-cell NHL that display a phenotype compatible with regulatory T cells (Treg cells). These cells express high levels of Foxp3 and CTLA-4, and are capable of suppressing the proliferation and cytokine production of autologous infiltrating CD4+CD25- T cells in B-cell NHL. Goal: To explore whether Treg cells exert a suppressive effect on the induction and function of autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. Results: Using fresh specimens obtained from patients with B-cell lymphoma, we found that intratumoral Treg cells had a significantly suppressive effect on autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. When activated CD8+ T-cells were cocultured with infiltrating CD4+CD25- T-cells, they displayed less proliferation than CD8+ T-cells cultured alone. However, we found that with a 1:4 ratio of stimulating cells (Treg cells) to responding cells (CD8+ T-cells), intratumoral Treg cells completely inhibited the proliferation of autologous tumor-infiltrating CD8+ T-cells activated with PHA. Furthermore, we have observed that 20% of infiltrating CD8+ T-cells in fresh isolated samples from B-cell NHL coexpress perforin and granzyme B. When intratumoral Treg cells were cocultured with CD8+ T-cells, the Treg cells inhibited the activation-induced production of perforin and granzyme B of autologous tumor-infiltrating CD8+ T-cells in a dose-dependent fashion. We also found that cytokine treatment (IL-2 and IL-12) or PHA activation induced the capability of tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells, which was accompanied by upregulation of expression of CD107a on the surface of cytotoxic CD8+ T-cells. Intratumoral Treg cells significantly inhibit cytotoxicity of activated infiltrating CD8+ T-cells to lymphoma B-cells. Finally, we found that there was an inverse correlation of cell frequencies between intratumoral Treg cells and tumor-infiltrating CD8+ T cells in patients with B-cell NHL, suggesting that Treg cells may inhibit the migration of cytotoxic T-cells to the sites of B-cell lymphoma. Conclusion: Intratumoral Treg cells dramatically suppress the proliferation and activation-induced production of granules in infiltrating CD8+ T-cells in B-cell NHL. These Treg cells also inhibit the ability of activated tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells in vitro, and may prevent the migration of CD8+ cells to the sites of lymphoma. Taken together, these data indicate an important role for intratumoral Treg cells in CTL-mediated anti-tumor immunity in B-cell NHL.

Description: Most non-Hodgkin lymphomas (NHLs) are of B-cell origin, but the tumor tissue can be variably infiltrated with T cells. In the present study, we have identified a subset of CD4+CD25+ T cells with high levels of CTLA-4 and Foxp3 (intratumoral Treg cells) that are overrepresented in biopsy specimens of B-cell NHL (median of 17% in lymphoma biopsies, 12% in inflammatory tonsil, and 6% in tumor-free lymph nodes; P = .001). We found that these CD4+CD25+ T cells suppressed the proliferation and cytokine (IFN-γ and IL-4) production of infiltrating CD4+CD25- T cells in response to PHA stimulation. PD-1 was found to be constitutively and exclusively expressed on a subset of infiltrating CD4+CD25- T cells, and B7-H1 could be induced on intratumoral CD4+CD25+ T cells in B-cell NHL. Anti-B7-H1 antibody or PD-1 fusion protein partly restored the proliferation of infiltrating CD4+CD25- T cells when cocultured with intratumoral Treg cells. Finally, we found that CCL22 secreted by lymphoma B cells is involved in the chemotaxis and migration of intratumoral Treg cells that express CCR4, but not CCR8. Taken together, our results suggest that Treg cells are highly represented in the area of B-cell NHL and that malignant B cells are involved in the recruitment of these cells into the area of lymphoma.

Description: Background: Non-Hodgkin lymphomas (NHL) are increasing in incidence and are now the fifth most common tumor diagnosed each year in the United States. Most NHLs are of B-cell origin but the tumor tissue is variably infiltrated with T-cells. Our group has shown in diffuse B-cell large cell lymphoma, that a high number of intratumoral CD4+ T-cells predicts a better overall survival. It has been shown that recently-characterized CD4+CD25+ regulatory T-cells (Treg cells) played an important role in the mediation of anti-tumor immunity. However, there is no data on the role of intratumoral Treg cells in suppression of autologous infiltrating CD4+ T-cells in B-cell NHL. Goal: To investigate the effect of intratumoral Treg cells on the proliferation of tumor-infiltrating CD4+CD25- T-cells, to determine the underlying mechanism of the T-cell suppression, and evaluate the role that malignant B-cells may play in the recruitment of Treg cells to the site of B-cell NHL. Results: We identified a subset of CD4+CD25+ T-cells over-represented in biopsy specimens of B-cell NHL (these cells comprise 17% of cells in lymphoma biopsies, compared 7% of peripheral blood mononuclear cells, 12% of cells in inflammatory tonsil and 6% of cells in tumor free lymph nodes; p-value =0.001). These CD4+CD25+ T-cells are memory-like T-cells (CD45RO+ and CD45RA−) and express high levels of CTLA-4 and Foxp3 when compared to autologous tumor-infiltrating CD4+CD25- T-cells. Importantly, these CD4+CD25+ T-cells displayed the ability to suppress the proliferation and cytokine (IFN-g and IL-4) production of tumor-infiltrating CD4+CD25- T-cells in response to PHA stimulation. Treatment with anti-B7-H1 antibody or PD-1 fusion protein enhanced the proliferation of infiltrating CD4+CD25- T-cells when co-cultured with intratumoral CD4+CD25+ T-cells. Our results suggest that interaction between B7-H1 and PD-1 accounts for about 30% of intratumoral Treg cell-mediated inhibition of autologous infiltrating CD4+CD25- T-cells in tumor sites of B-cell NHL. Lastly, we found that CCL22 secreted by lymphoma B-cells is involved in the chemotaxis and migration of intratumoral CD4+CD25+ T-cells which express chemokine receptor CCR4, but not CCR8. Conclusion: Our results suggest that tumor microenvironmental CD4+CD25+ regulatory T-cells are important regulators of tumor immunity and that these cells are recruited to the area of lymphoma involvement by the malignant B-cells.

Description: Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19+ purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.

Description: Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma characterized by rapid disease progression. Although the clinical outcome of MCL patients has improved in the recent years, relapses and progressive emergence of resistance to treatment are common. Therefore, alternative therapeutic strategies are in great need for the treatment of MCL patients. Polycomb repressive complex 2 (PRC2), the mammalian enzymatic complex plays crucial roles in regulation of normal and malignant hematopoiesis. Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of PRC2 complex is frequently amplified or overexpressed in most solid tumor types. However, EZH2 has been reported to harbor mutations of tyrosine 641 to asparagine (Y641N) in 22% of GCB diffuse large B-cell lymphoma (DLBCL) and 7% of follicular lymphoma (FL), but not in MCL. In the current study, we sought to elucidate the role of PRC2 in MCL and its implications for epigenetic therapy. We first assessed the expression of PRC2 components EZH2, SUZ12 and EED in several MCL cell lines (Jeko, Mino, Granta, JVM2 and Z138) and normal B-cells. Western blot analysis demonstrated that SUZ12 and EED along with EZH2 are overexpressed in proliferating MCL cell lines but not in normal B-cells. Furthermore, aberrant expression of H3 trimethylation (H3K27me3), di-methylation (H3K27me2) and mono-methylation (H3K27me1) was observed in all five MCL cell lines tested as compared to normal B cells. In order to understand if MCL cells have stable and enzymatically active PRC2 methyltransferase complex, we performed co-immunoprecipitation assay using EZH2 antibody and IgG as control. Significant amount of EED and SUZ12 enrichment was observed in the EZH2 immunoprecipitates from Granta and Mino MCL cells. Collectively this data demonstrates that in the MCL cells, PRC2 complex is aberrantly active and is associated with EZH2 upregulation. To study the role of EZH2 and EED in MCL cells, we generated expression constructs of the full-length and deletion fragments EZH2-F (full length EGH2), EZH2-del (EZH2 with deletion of SET domain), EED-F (full length EED) and EED-del (EED with deletion of WD40 domain), respectively. Interestingly, overexpression of EEDWT and EZH2WT led to a significantly (p=0.05) increased cell proliferation resulted in increased H3K27 trimethylation as compared to cells transfected with empty vector. Co-transfection with EZH2-del (SET domain) with the EZH2-F attenuated EZH2-F induced H3K27me3 and cell proliferation, suggesting oncogenic role of EZH2 enzymatic activity. Interestingly, co-transfection of EED-del (deficient in WD40 domain) had no effect on EED-F induced H3K27Me3, and the proliferation was less pronounced as compared with EED-F alone. EZH2 gene inhibition with EZH2-shRNA inhibited the growth of the MCL cells. Pharmacological inhibition of global methyltransferase activity by DZNep or EZH2 specific inhibitor GSK343 greatly suppressed the H3K27me3 level with little effect on H3K27me1 in Granta cells. However, treatment with GSK343 (but not DZNep) inhibited interaction between EZH2 and EED. Furthermore, treatment with EZH2 inhibitor, GSK343, produced significant growth inhibition (more than 90%) at 20uM concentration in Mino, Granta and Z138 cells. Taken together, these results suggest wild-type EZH2 is an epigenetic target for MCL, and MCL cells are highly sensitive to the EZH2 inhibition regardless of EZH2 mutations. Our findings indicate the use of PRC2 and EZH2 epigenetic inhibitors for the treatment of MCL patients. Overall, this study implies that overexpression of wild-type EZH2 has an oncogenic role in MCL, and suggest that wild-type EZH2 may confers similar active PRC2 addiction as mutant-EZH2 does in DLBCLs, FLs and other malignancies. Disclosures No relevant conflicts of interest to declare.

Description: The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

Description: 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.