Lymphoma Pre-clinical Models Research Articles

Preclinical evaluation of the BET bromodomain inhibitor BAY 1238097 for the treatment of lymphoma.

The epigenome is often deregulated in cancer and treatment with inhibitors of bromodomain and extra-terminal proteins, the readers of epigenetic acetylation marks, represents a novel therapeutic approach. Here, we have characterized the anti-tumour activity of the novel bromodomain and extra-terminal (BET) inhibitor BAY 1238097 in preclinical lymphoma models. BAY 1238097 showed anti-proliferative activity in a large panel of lymphoma-derived cell lines, with a median 50% inhibitory concentration between 70 and 208nmol/l. The compound showed strong anti-tumour efficacy invivo as a single agent in two diffuse large B cell lymphoma models. Gene expression profiling showed BAY 1238097 targeted the NFKB/TLR/JAK/STAT signalling pathways, MYC and E2F1-regulated genes, cell cycle regulation and chromatin structure. The gene expression profiling signatures also highly overlapped with the signatures obtained with other BET Bromodomain inhibitors and partially overlapped with HDAC-inhibitors, mTOR inhibitors and demethylating agents. Notably, BAY 1238097 presented invitro synergism with EZH2, mTOR and BTK inhibitors. In conclusion, the BET inhibitor BAY 1238097 presented promising anti-lymphoma preclinical activity invitro and invivo, mediated by the interference with biological processes driving the lymphoma cells. Our data also indicate the use of combination schemes targeting EZH2, mTOR and BTK alongside BET bromodomains.

L-mind: MOR208 combined with lenalidomide (LEN) in patients with relapsed or refractory diffuse large b-cell lymphoma (R-R DLBCL)—A single-arm phase II study.

7514 Background: The Fc-enhanced CD19 antibody MOR208 and the immunomodulatory drug LEN have demonstrated single agent activity in patients (pts) with R-R DLBCL. MOR208 and LEN have shown synergy in vitro and in vivo in preclinical lymphoma models. This ongoing phase II study assesses the safety and efficacy of MOR208 + LEN in pts with R-R DLBCL. Methods: Pts >18 years old with R-R DLBCL, ECOG 0–2, adequate organ function, having previously received ≥1 but not more than 3 prior therapies, including ≥1 CD20-targeting regimen and who are not candidates for autologous stem cell transplant (ASCT), are eligible. Treatment comprises up to 12, 28-day (d) cycles (C) of MOR208 12 mg/kg IV, weekly during C1–3 (loading dose d4 of C1); every second week C4–12 + LEN 25 mg po d1–21, C1–12. Pts progression-free after 12 cycles receive up to 12 additional cycles of MOR208 (every second week). The primary endpoint is the overall response rate (ORR) by central radiology assessment. Secondary endpoints include disease control, duration of response, progression-free and overall survival, safety, and response by cell of origin and other biomarkers. A preplanned safety evaluation was undertaken. Results: 31 of 80 planned pts were enrolled prior to data cutoff (3 January 2017). Median age was 74 years (range 47–82); 45% of pts received ≥2 prior lines of therapy; 23% had rituximab refractory disease; 74% had Ann Arbor stage ≥III disease; 65% had elevated lactate dehydrogenase level, and 52% had a poor revised International Prognostic Index (3-5). The most common treatment-emergent adverse events (any grade/grade ≥3 [% pts]) were neutropenia (39/26), anemia (23/0) thrombocytopenia (16/6), infections (26/10) diarrhea (13/0), pyrexia (13/0), and rashes (13/6). Of 26 response evaluable pts (median follow-up 3.3 months), ORR (investigator assessed) was 58% (15 pts), with 7 (27%) complete responses. Median time to response was 1.8 months. Conclusions: The combination of MOR208 + LEN is well tolerated and shows promising activity in pts with R-R DLBCL. Accrual and follow-up of pts is ongoing, as are cell of origin and other biomarker analyses. Clinical trial information: NCT02399085.

MiR-28 regulates the germinal center reaction and blocks tumor growth in preclinical models of non-Hodgkin lymphoma

AbstractNon-Hodgkin lymphoma comprises a variety of neoplasms, many of which arise from germinal center (GC)-experienced B cells. microRNA-28 (miR-28) is a GC-specific miRNA whose expression is lost in numerous mature B-cell neoplasms. Here we show that miR-28 regulates the GC reaction in primary B cells by impairing class switch recombination and memory B and plasma cell differentiation. Deep quantitative proteomics combined with transcriptome analysis identified miR-28 targets involved in cell-cycle and B-cell receptor signaling. Accordingly, we found that miR-28 expression diminished proliferation in primary and lymphoma cells in vitro. Importantly, miR-28 reexpression in human Burkitt (BL) and diffuse large B-cell lymphoma (DLBCL) xenografts blocked tumor growth, both when delivered in viral vectors or as synthetic, clinically amenable, molecules. Further, the antitumoral effect of miR-28 is conserved in a primary murine in vivo model of BL. Thus, miR-28 replacement is uncovered as a novel therapeutic strategy for DLBCL and BL treatment.

Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

BackgroundPharmacological inhibition of B cell receptor (BCR) signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton’s kinase (Btk) and spleen tyrosine kinase (Syk) inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents.MethodsWe evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn), in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma.ResultsIQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest.ConclusionsThese results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors

ABSTRACTThe use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use.

Identification of Anti-Lymphoma Biomarkers of Response to the Anti-CD37 Antibody Drug Conjugate (ADC) IMGN529

Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response.Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down).Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P<0.001) in B cell lymphoma cell lines (n= 46; median IC50=450pM; 95%C.I., 150-800pM) than in T cell lymphoma cell lines (n=8; median IC50=22.5nM; 95%C.I., 14-40nM). The median IC50 for DM1 was 30pM (C.I.95%, 20-40pM) with no differences between B and T cell lymphoma origin. IMGN529 induced apoptosis in 33/54 (61%) lymphoma cell lines. Surface CD37 expression was higher in cell lines derived from B than from T cells (P< 0.0001): IMGN529 IC50 values, but not of DM1, were negatively correlated with surface CD37 expression across all cell lines (R=-0.39; P= 0.018), but not within the individual B or T cell subgroups. Among B cell lines, DLBCL cell of origin, TP53 status or the presence of BCL2 translocation did not affect the sensitivity to IMGN529, while IC50s were higher in the presence of MYC translocation (P= 0.043). No association was seen between IMGN529-induced apoptosis or the sensitivity to DM1 with DLBCL cell of origin, TP53 status or the presence of BCL2 or MYC translocations.We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC.Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. DisclosuresRossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.

Anti-CD19 ARTEMISTM Therapy Drastically Reduces Cytokine Release without Compromising Efficacy Against Preclinical Lymphoma Models

Anti-CD19 chimeric antigen receptor (CAR) T cell therapies for B cell malignancies have demonstrated the remarkable curative potential of T cell immunotherapies. However, in clinical trials anti-CD19-CAR T cells continue to trigger life threatening adverse events that are often associated with excessive cytokine release and excessive T-cell proliferation. We reasoned that the activation pathway of current CAR T cells could be altered to better regulate proliferation and cytokine secretion, and thus disentangle the correlation between cytokine release syndrome (CRS) and efficacy of T cell-based therapies.Through protein engineering, we developed the ARTEMISTM (1) signaling platform which when expressed on primary T-cells results in a dramatic reduction of cytokine release during tumor cell lysis, without sacrificing efficacy. Using a human phage display library, we also identified several human CD19 antibodies with improved specificity and affinity that will be less immunogenic as compared to the murine-derived anti-CD19 antibodies that are currently used in most trials. Our lead antibody clone CD19(7) was then engineered into both CD28z-CAR and ARTEMISTM platforms for comparison.When tested in vitro, both CD19(7)-ARTEMISTM T cells and CD19(7)-CD28z-CAR T cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour killing assays, ARTEMIS™ T cells secreted over 1000-fold less IL-2 and dramatically lower levels of IFN-γ, GM-CSF, IL-10 and IL-6. ARTEMISTM T cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion. Furthermore, during in vitro T cell expansion, ARTEMISTM cells were enriched for naïve/central memory subpopulations, had lower expression of granzyme B, a marker of terminal differentiation, and had reduced rates of receptor internalization upon antigen engagement. These characteristics suggest that T-cells activated through the ARTEMISTM receptor will have improved persistence and long-term proliferation potential, as well as a safer, more controlled cytokine release when used for T-cell therapies.When tested in vivo against CD19+ Raji systematic lymphoma xenografts, intravenous administration of CD19(7)-ARTEMISTM T cells caused rapid, complete, and lasting tumor regression that was better than that achieved with an equal dose of CD19(7)-CD28z-CAR T cells (Figure 1). In agreement with our in vitro data, mice treated with ARTEMISTM T cells had nearly undetectable levels of cytokines in their blood at 24 hours post dosing, a time in which CD19(7)-CAR-treated mice had markedly elevated levels of human IFN-γ, IL-2, TNFα, and IL-10.While flow cytometry analysis of the peripheral blood showed that CD19(7)-CAR T cells expanded more rapidly in mice, CD19(7)-ARTEMISTM T cells better controlled Raji tumor growth and were negative for PD-1 expression which was high on circulating CAR T cells. At 7 weeks post dosing, a time when all ARTEMISTM T cell-treated mice had no detectable tumors, they were re-challenged with Raji lymphoma. While tumors grew rapidly in control mice, ARTEMISTM T cell-treated mice resisted the Raji lymphoma re-challenge, indicating that ARTEMISTM T cells persisted in these mice despite the absence of tumors and remained antigen-responsive (Figure 2).Our data demonstrates that CD19(7)-ARTEMISTM T cells are highly potent against lymphoma preclinical models while releasing drastically lower levels of cytokines. Thus we have developed and pre-clinically validated a novel fully human anti-CD19 T cell therapy that has the potential to persist longer in patients and, importantly, presents a lower risk of cytokine-related toxicities without compromising efficacy. A clinical trial testing CD19(7)-ARTEMISTM T cell therapy in humans is expected to begin in 2017. [Display omitted] [Display omitted] DisclosuresLiu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties. Long:Eureka Therapeutics: Employment, Equity Ownership. Green:Eureka Therapeutics: Employment. Horan:Eureka Therapeutics: Employment. Zimdahl:Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership, Patents & Royalties.

JQ1, a c-MYC Inhibitor Targets Hexokinase II and Enhances the Cytotoxic Effects of Chemotherapeutic Agents in Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Results from thecollaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated a poor clinical among patients treated with second-line chemo-immunotherapy in the post-rituximab era, stressing the need to identify and/or optimize novel targeted agents. C-myc expression was found correlate with a poor clinical outcome in patients with newly diagnosed or relapsed/refractory DLBCL. Previously, we found that JQ1 overcomes rituximab-chemotherapy resistance in lymphoma pre-clinical models. JQ1 exhibited synergistic activity when combined with chemotherapy agents. We also demonstrated that hexokinase II was highly expressed in rituximab resistant cell lines (RRCL), and its expression was associated with a deregulation in the glucose metabolism and an increase in the apoptotic threshold leading to chemotherapy resistance. In our current work, we evaluated molecular studies dissecting the cellular pathways affected by JQ1.Methods: A panel of rituximab-sensitive (RSCL) and RRCL cell lines were exposed to escalating doses of JQ1 (0-8μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of mitochondrial membrane potential (Δψm) following JQ1 exposure was assessed by DiOC6 staining. Changes in c-MYC, HKII, HKI, Voltage dependent anion channel protein (VDAC) and p21 expression levels were determined by western blotting. HKII was silencing using siRNA interference in RRCL (Raji-4RH) and changes in HKII, HKI, VDAC, c-MYC and p21 protein expression was determined. HKII knockdown or scramble Raji-4RH cells were exposed to different chemotherapy agents and JQ1; cell viability and mitochondrial membrane potential was accessed.Result:JQ1 induced a dose-and time- dependent cell death in cell lines. In vitro exposure cells to JQ1 resulted in a loss of mitochondrial potential and induced G1 cell cycle arrest. In vitro exposure to JQ1 decreased c-MYC and HKII expression levels and induced of p21 expression. Complete silencing of HKII re-sensitized cell to chemotherapy (doxorubicin, vincristine and bortezomib). On the other hand, HKII knockout abrogated JQ1 anti-tumor activity.Conclusion: Our data suggests that JQ1 had anti-tumor activity against rituximab-sensitive or resistant pre-clinical models. The inhibition of HKII following JQ1 exposure may contribute to lower the mitochondrial membrane potential and enhance the chemotherapeutic effect. Our finding highlights a better understanding in the molecular events triggered by JQ1 as a potential role in the treatment of B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Targeting BET Bromodomains in Pre-Clinical Models of Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway.Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values.Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI<0.9) at multiple dose combinations while other combinations exhibited primarily additive effects.Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. DisclosuresNo relevant conflicts of interest to declare.

CD47 Monoclonal Antibody SRF231 Is a Potent Inducer of Macrophage-Mediated Tumor Cell Phagocytosis and Reduces Tumor Burden in Murine Models of Hematologic Malignancies

The transmembrane protein CD47 is an immunoglobulin superfamily member involved in multiple cellular processes, including cell migration, adhesion and T cell function. The interaction between CD47 and signal regulatory protein alpha (SIRPα), an inhibitory protein expressed on macrophages, prevents phagocytosis of CD47-expressing cells. CD47 was originally identified as a tumor antigen on human ovarian cancer and was subsequently shown to be expressed on multiple human tumor types, including both hematologic and solid tumors. In many hematologic cancers, high CD47 expression is associated with poor clinical outcomes. CD47 is also expressed at low levels on virtually all non-malignant cells, and loss of expression or changes in membrane distribution may serve as markers of aged or damaged cells, particularly on red blood cells (RBC). High expression of CD47 on tumors blocks phagocytic uptake, subsequent antigen cross-presentation and T cell activation, which collectively contribute to tumor immune evasion. Agents that block the CD47-SIRPα interaction can restore phagocytic uptake of CD47+ target cells and lower the threshold for macrophage activation, which can enhance the efficacy of therapeutic antibodies with ADCC-enabling activity. We developed and characterized a CD47 blocking antibody and evaluated its activity in multiple hematologic models.SRF231 is a fully human monoclonal antibody that binds with high affinity to human CD47 and blocks the CD47-SIRP□ interaction. SRF231 promotes macrophage-mediated phagocytic clearance of several hematologic primary tumor samples and cell lines in vitro. For example, SRF231 increases phagocytosis of Raji tumor cell line targets with an EC50 of ~300 ng/ml. Enhanced phagocytosis is preferential for tumor cells over normal leukocytes and RBC. Tumor cell phagocytosis can be enhanced in the presence of opsonizing antibodies (e.g., anti-CD20 Ab) when co-administered with SRF231.In vivo efficacy of SRF231 was assessed in several preclinical murine xenograft models of hematologic malignancies. Notably, SRF231 administration led to profound tumor growth inhibition in models of multiple myeloma, diffuse large B cell lymphoma and Burkitt's lymphoma as a single agent and in combination with opsonizing antibodies. In the Raji xenograft model, single agent therapy leads to 112% tumor growth inhibition. This anti-tumor activity is at least partially dependent on macrophages, as depletion of macrophages via clodronate administration leads to reduced tumor growth inhibition. Tumor-associated macrophage (TAM) numbers and polarization status are also modulated by SRF231 treatment.In summary, the CD47 mAb SRF231 induces robust tumor cell phagocytosis and tumor clearance both alone and in combination with opsonizing antibodies in pre-clinical models of myeloma and lymphoma. These properties warrant further development of SRF231 in hematologic malignancies. SRF231 is currently in IND-enabling studies and is expected to enter clinical trials in 2017. DisclosuresNo relevant conflicts of interest to declare.

Denintuzumab Mafodotin Stimulates Immune Responses and Synergizes with CD20 Antibodies to Heighten Anti-Tumor Activity in Preclinical Models of Non-Hodgkin Lymphoma

Denintuzumab Mafodotin (SGN-CD19A) is an antibody-drug conjugate (ADC) composed of an anti-CD19 antibody attached to a synthetic cytotoxic agent, monomethyl auristatin F (MMAF). The ADC binds CD19, internalizes, and releases cys-mcMMAF, which ultimately results in G2-M growth arrest and induction of apoptosis in targeted cells (Law et. al. AACR 2011). SGN-CD19A has shown convincing antitumor activity in relapsed/refractory non-Hodgkin lymphoma patients, resulting in a significant number of objective responses, including complete responses with prolonged durability (Moskowitz et. al. Blood 2015). To further characterize the anti-tumor activity of SGN-CD19A, we evaluated whether immune-mediated cell killing and immunogenic cell death (ICD) act in addition to the previously described apoptotic cell death. We then examined whether these mechanisms of cell death are augmented by clinically approved CD20 antibodies.Our results show that SGN-CD19A supports immune-mediated killing of lymphoma cell lines in vitro through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Assessment of disseminated and subcutaneous tumor xenografts showed that SGN-CD19A activity is reduced when the wild type antibody is replaced with IgG variants that lack effector function. We also found depletion of macrophage or NK cells eliminates the improved survival conferred by the parental antibody of SGN-CD19A. Together these data provide strong evidence that effector function contributes to activity of SGN-CD19A in vivo.Next, we implemented an in vitro system pairing lymphoblastoid cell lines (LCLs) with autologous PBMC to investigate the effect of SGN-CD19A-mediated cell death on immune activation. Treatment of LCL with SGN-CD19A drove hallmarks of ICD as indicated by activation of the ER stress response along with upregulation of surface calreticulin. In vitro co-culture of SGN-CD19A treated LCL with autologous PBMC resulted in heightened monocyte activation and expansion of LCL-reactive cytotoxic T cells supporting a role for SGN-CD19A in augmenting anti-tumor immune responses.Finally, we evaluated the anti-tumor activity of SGN-CD19A in the presence and absence of clinically relevant CD20 antibodies. Results showed that CD20-targeted antibodies impact internalization of CD19 antigen leading to improved drug delivery of SGN-CD19A. Blocking B cell signaling inhibits the effect of CD20 antibodies on CD19 and SGN-CD19A. The improved anti-tumor activity observed with SGN-CD19A plus CD20 therapeutics was confirmed in vivo using rituximab. In conclusion, our results demonstrate SGN-CD19A is a multifunctional ADC capable of eliciting cell killing through immune-mediated mechanisms as well as delivery of the cytotoxic payload, cys-mcMMAF. Importantly, the described mechanisms of SGN-CD19A activity are further augmented by CD20 antibodies. These studies support the ongoing clinical evaluation of SGN-CD19A, which is currently being tested in randomized Phase II clinical trials in combination with R-ICE (NCT02592876) as well as R-CHOP (NCT02855359). DisclosuresVan Epps:Seattle Genetics: Employment. Heiser:Seattle Genetics: Employment. Cao:Seattle Genetics: Employment. Klussman:Seattle Genetics: Employment. Yu:Seattle Genetics: Employment. Stone:Seattle Genetics: Employment. Neale:Seattle Genetics: Employment. Gardai:Seattle Genetics: Employment. Law:Seattle Genetics, Inc.: Employment, Equity Ownership. Ryan:Seattle Genetics: Employment.

BET inhibitors in the treatment of hematologic malignancies: current insights and future prospects

The bromodomain and extra-terminal (BET) family of proteins are important epigenetic regulators involved in promoting gene expression of critical oncogenes. BET inhibitors have been demonstrated to repress c-Myc expression, and were initially shown to have efficacy in a number of c-Myc-dependent hematologic malignancies. Recent studies have now revealed a broader role for BET inhibitors in hematologic malignancies. In this review, we summarize the efficacy of BET inhibitors in preclinical models of acute leukemia, lymphoma, and multiple myeloma. We also summarize recent results of clinical trials utilizing BET inhibitors in hematologic malignancies, characterize potential resistance mechanisms to BET inhibitors, and discuss potential combination therapies with BET inhibitors in patients with hematologic malignancies.

Combination of the MEK inhibitor pimasertib with BTK or PI3K-delta inhibitors is active in preclinical models of aggressive lymphomas

Lymphomas are among the most common human cancers and represent the cause of death for still too many patients. The B-cell receptor with its downstream signaling pathways represents an important therapeutic target for B-cell lymphomas. Here, we evaluated the activity of the MEK1/2 inhibitor pimasertib as single agent and in combination with other targeted drugs in lymphoma preclinical models. Cell lines derived mature B-cell lymphomas were exposed to increasing doses of pimasertib alone. Immunoblotting and gene expression profiling were performed. Combination of pimasertib with idelalisib or ibrutinib was assessed. Pimasertib as single agent exerted a dose-dependent antitumor activity across a panel of 23 lymphoma cell lines, although at concentrations higher than reported for solid tumors. Strong synergism was observed with pimasertib combined with the PI3K inhibitor idelalisib and the BTK inhibitor ibrutinib in cell lines derived from diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma. The data were confirmed in an in vivo experiment treating DLBCL xenografts with pimasertib and ibrutinib. The data presented here provide the basis for further investigation of regimens including pimasertib in relapsed and refractory lymphomas.

Abstract B31: EZH2 suppresses the interferon-stimulated gene response in non-Hodgkin lymphoma

Abstract Pharmacological inhibition of the histone lysine methyltransferase EZH2 has emerged as a therapeutic strategy for the treatment of non-Hodgkin Lymphoma (NHL), in particular for cases with mono-allelic mutations in the EZH2 catalytic domain. Potent, selective EZH2 small molecule inhibitors have achieved tumor regression in mutant EZH2-containing preclinical lymphoma models and several of these inhibitors are currently engaged in cancer-focused clinical trials. Here, we show that the presence of EZH2 mutations do not always confer EZH2 inhibitor sensitivity. We discovered that EZH2 is usurped by lymphoma cells to attenuate JAK/STAT signaling and to repress pro-apoptotic interferon response genes. EZH2 inhibition results in the broad induction of interferon-stimulated genes (ISGs) in several phenotypically sensitive NHL cell models. In mutant EZH2-containing insensitive models, EZH2 inhibitors synergize specifically with type I interferons (IFNs) in vitro and in vivo to induce ISGs and apoptosis. The profound combinatorial activity of EZH2 inhibitors and type I IFNs is not restricted to NHL models with mutant EZH2 and, is preferentially observed in models that are not affected by either single agent. Molecular consequences of EZH2 inhibitor and type I IFN combinatorial treatment include STAT1 activation, binding of STAT transcription factor complexes to ISGs, reduction of H3K27me3 levels and increase of H3K4me3 levels at ISG transcriptional start sites, and ISG transcriptional activation. The molecular and anti-proliferative effects of the combination can be suppressed by inactivation of the JAK/STAT signaling pathway, by using the JAK inhibitor ruxolitinib. We suggest that EZH2 inhibitors can be therapeutically combined with type I IFNs for the treatment of NHL. Citation Format: William D. Bradley, Chih-Chi Yuan, Feng Zhao, Christina Lee, Kaylyn E. Williamson, Shilpi Arora, Charlie Hatton, Barbara M. Bryant, Patrick Trojer. EZH2 suppresses the interferon-stimulated gene response in non-Hodgkin lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B31.

Translational Focus on Targeting HDAC6 with Ricolinostat Confirms Potent Activity in Preclinical Models of Lymphoma and a Favorable Toxicity Profile in Patients with Relapsed or Refractory Lymphoma

Pan-class HDAC inhibitors are active agents for select subtypes of lymphoma but are often associated with toxicities such as fatigue, diarrhea/constipation, cardiac arrhythmias and myelosuppression. HDAC6 plays a critical role in protein homeostasis by managing misfolded proteins and inducing the unfolded protein response (UPR). Ricolinostat is an isoform selective HDAC6 inhibitor. We evaluated the preclinical effects of ricolinostat and translated the findings into a multicenter Phase 1b study in patients with lymphoma (NCT 02091063). Cytotoxicity was evaluated in 16 lymphoma cell lines (8 DBCL, 4 MCL, 4 TCL) by Cell TiterGlo and Annexin V. Ricolinostat has activity with IC50 values ranging from 0.9-4.7 uM range, which is in the range observed in clinical PK studies. Ricolinostat displayed marked synergy with bortezomib, PI3K inhibitors, disatinib, alisertib, and ibrutinib with synergy coeficients of 0.24, 0.29, 0.64, 0.07, 0.16 respectively (values In an effort to translate these findings, a multi-center Phase Ib open-label, single agent study designed to study the safety of 2 dose schedules of ricolinostat in patients with relapsed or refractory lymphoid malignancies was initiated. Patients were treated with 160 mg of oral ricolinostat continuously on a 28 day cycle. Patients were accrued sequentially to 2 dose cohorts: 3 patients were enrolled on single daily dosing (QD) schedule, and following safety assessment, patients were enrolled to twice daily dosing (BID). Samples were collected for PK and PD analysis. As of July 25th 2015, 11 patients were enrolled on the phase Ib study and were evaluated for safety. The median age was 36 yrs (24-86) and 7 (64%) were male. Sixty-four percent had 5 prior treatments (range: 1-15). Histologies included HL (N=3) and NHL (N=8) including DLBCL=3, MZL=2, FL=1, PTCL=1, PTLD=1. Three patients were enrolled in QD dosing and 8 patients in the BID schedule. There were no DLTs at either of the dosing schedules, and the maximum administrable dose was determined on the BID dosing schedule. The median number of cycles completed was 2 and ranged from 1 through 12+ (currently ongoing). The only grade 3 toxicities reported were anemia (27%), hyponatremia (9%), hypercalcemia (9%) and hypotension (9%), none thought to be attributed to the study drug. Other treatment emergent events included nausea (45%), diarrhea (36%), fatigue (36%) and vomiting (27%) which were all grade 1 and 2. Three patients achieved stable disease, all of whom were treated on the BID schedule (3 of 8, 37.5%), no complete or partial responses have been observed to date. One patient with refractory HL with 14 prior lines of therapy achieved stable disease (SD) and had symptomatic improvement corresponding to an increase of hemoglobin after 9 cycles, as well as resolution of B symptoms. Another patient with PTCL had immediate resolution of B symptoms such as fevers and pruritus after starting QD dosing. This is the first reported experience with an isoform selective HDAC inhibitor to be studied for the treatment of patients with relapsed or refractory lymphoid malignancies. Preclinical studies support the notion that ricolinostat mediates its effects through the UPR and potently synergizes with complementary agents. Ricolinostat is well tolerated with no DLTs. Thirty-seven percent of patients achieved SD on the BID schedule. PK and PD data will be evaluated for acetylation of alpha-tubulin and modulation of the UPR in an effort to confirm preclinical findings. Efficacy will be evaluated in a phase II study. We believe that ricolinostat in rational combinations will lead to effective treatment for patients with relapsed lymphoid malignancies. Disclosures Amengual:Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. Off Label Use: Ricolinostat is being studied for the treatment of lymphoma. Neylon:Seattle Genetics: Speakers Bureau; Celgene: Speakers Bureau; Genentech: Speakers Bureau; Gilead: Speakers Bureau. Deng:TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Sawas:Seattle Genetics: Research Funding; Gilead Sciences: Honoraria. Shah:Pharmacyclics: Speakers Bureau; NCCN: Consultancy; SWOG: Consultancy; Rosetta Genomics: Research Funding; Acetylon Pharmaceuticals, INC: Membership on an entity9s Board of Directors or advisory committees; Plexus Communications: Honoraria; Spectrum: Speakers Bureau; Bayer: Honoraria; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; DeBartolo Institute for Personlaized Medicine: Research Funding; Seattle Genetics: Research Funding; Janssen: Speakers Bureau. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. O9Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Acetylon Pharmaceuticals, INC: Consultancy; Mundipharma: Consultancy, Honoraria, Research Funding.

Dual Inhibition of PKC Delta By Carfilzomib (CFZ) and Ibrutinib Results in Synergistic Activity in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models and in Primary Tumor Cells Isolated from B-Cell Lymphoma Patients

Introduction: A better understanding in the role of the B-cell receptor-signaling pathway in lymphomagenesis led to the clinical development of ibrutinib is an orally bioavailable Bruton's tyrosine kinase inhibitor (BTKi). Ibrutinib has clinical activity in several subtypes of B-cell lymphoma (follicular lymphoma, mantle cell lymphoma and certain diffuse large B-cell lymphoma [DLBCL] subtypes). Recently, an increasing number of ibrutinib-treated patients are developing disease progression, stressing the need to optimize its therapeutic potential. To this end, we evaluated the biological effects and interactions of ibrutinib and CFZ, a non-reversible proteasome inhibitor in several lymphoma pre-clinical models including primary tumor cells isolated from lymphoma patients.Methods: A panel of rituximab-sensitive (RSCL) and rituximab-resistant (RRCL) cell lines as well as primary tumor cells isolated from B-cell lymphoma patients (N=20) were exposed to ibrutinib and/or CFZ. Changes in cell viability were evaluated by Presto Blue and Titer Glo assays respectively. Coefficient of synergy was determined using the CalcuSyn software. To further study the mechanisms responsible for the biological effects of ibrutinib and CFZ, RSCL and RRCL were exposed to Ibrutinib+/- CFZ for 48hr. Changes in apoptosis, mitochondrial potential, and cell cycle distribution were evaluated by flow cytometry by AnnexinV-PI, DiOC6 or PI staining respectively. Subsequently, protein lysates were isolated from ibrutinib+/- CFZ exposed RSCL or RRCL and changes in downstream targets (PKCδ a member of diacylglycerol-dependent, calcium-independent serine/threonine-specific protein kinases PKC isoform know to be a substrate of caspase 3 and able to mediate apoptosis), Bcl-2 family members and cell cycle regulatory proteins were evaluated by Western blotting.Results: In vitro exposure of RSCL, RRCL, and primary tumor cells to ibrutinib and CFZ resulted in synergistic anti-tumor activity. In addition, the combination of ibrutinib and CFZ resulted in a more pronounced decrease in the mitochondrial potential, higher degree of apoptosis (as demonstrated by flow cytometry and caspase 3 or PARP cleavage) and increase in G1 cell cycle arrest when compared to single agent ibrutinib or CFZ. Perhaps related to these findings, we found that exposure of lymphoma cells to Ibrutinib and CFZ resulted in an increase in caspase 3, the active form of PKCδ and BAX levels. Using caspase inhibitor Q-VD-OPh rescued the combination killing effect of ibrutinib and CFZ, as well as abrogated induction of the active form of PKCδ. In addition, Ibrutinib in combination with CFZ decreased cyclin E and CDK2 levels.Conclusions: Together our data suggests that ibrutinib and CFZ had a strong synergistic anti-tumor activity against rituximab-chemotherapy sensitive and resistant lymphoma cells and patient samples. At the molecular level, the combination of ibrutinib and CFZ affects the balance of the apoptotic machinery and key regulatory proteins of the cell cycle that could play an important role in the synergistic effects observed. Further signaling and in vivo studies are necessary to evaluate the therapeutic role of ibrutinib in combination with carfilzomib in B-cell lymphoma (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Funds). DisclosuresCzuczman:MorphoSys: Consultancy; Celgene: Employment; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.

Mechanisms of Acquired Drug Resistance to the Isoform Selective HDAC6 Inhibitor Ricolinostat Reveals Markedly Upregulated Elements of the BTK Pathway Revealing Rational Drug : Drug Combinations

HDAC6 is cytoplasmic with non-histone substrates and binds poly-ubiquinated misfolded proteins facilitating transport to the aggresome for proteasome-independent degradation. The aggresome is a key outlet for the unfolded protein response (UPR), a quality control mechanism that promotes survival during endoplasmic reticulum stress and signals CHOP (C/EBP-homologous protein) mediated apoptosis when protein homeostasis cannot be reestablished. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed lymphoma cell lines resistant to the selective HDAC6 inhibitor ricolinostat (Acetylon Pharmaceuticals, Inc). Ricolinostat has demonstrated activity in preclinical models of multiple myeloma and lymphoma and is currently being studied in the clinical setting for both of these diseases (NCT02091063, NCT01323751, NCT01997840, NCT01583283).The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ricolinostat over 1 yr. Systematic incremental increase in drug exposure led to the development of distinct cell lines with IC50 values 10-20 fold greater than that for parental lines (P). The resistant R10-OCI-LY10 (R10) achieved an IC50 of 10 uM and the R20-OCI-LY10 (R20) achieved an IC50 of 20 uM compared to P's IC50 of 0.9 uM at 48 hours. Resistance was maintained after repeated passages for ≥1 month and was not overcome by inhibition of efflux pumps as determined with verapamil co-exposure. Compared to P, R10 and R20 did not depolarize the mitochondrial membrane following treatment with ricolinostat 2.5 µM. However, there was no difference in the BH3 profiles of the R- and P-cell lines. Interestingly both R10 and R20 were also resistant to vorinostat which strongly inhibits HDAC6 (R20 IC50=7 uM vs P IC50=2 uM), and sensitive to romidepsin which is known to predominantly inhibit HDAC1, 2 and 3 with minimal activity against HDAC6 (R20 IC50=5.2 uM vs P IC50=4.5 uM). In addition, treatment of R20 with bortezomib demonstrated intermediate activity underscoring the idea that although the drug targets are vastly different, both drug mechanisms converge on the processing of misfolded proteins (R20 IC50=8 nM vs P IC50=3.3 nM). Accordingly, R10 and R20 displayed down-regulation of GRP78, the master regulator of the UPR, with concomitant up-regulation of both the PERK and IRE-1 pathways leading to up-regulation of p-eIF2a, ATF4, MAPK10 and XBP-1. Additionally, AKT was more highly phosphorylated in R10 and R20 compared to P.Gene expression profiling was performed on R10 and R20 independently and compared to P. Results were analyzed by GSEA and gene cluster analysis. There were 1363 genes in common from R10 and R20 2-log fold up-regulated as compared to P and 1825 genes in common from R10 and R20 2-log fold down-regulated as compared to P (p< 0.05). In addition to changes in the UPR, differentially expressed genes in R10 and R20 included up-regulation of MAPK10, HELIOS, HDAC9 and FYN, as well as down-regulation of SH3BP5 and LCK. The change in expression was confirmed by PCR and western blot analysis. Given the up-regulation of FYN, a tyrosine kinase in the B-cell receptor pathway, and down-regulation of SH3BP5 a negative regulator of the Bruton's Tyrosine Kinase, R10 and R20 cells were treated with ibrutinib 0.1 uM which was able to overcome resistance with 70% viability in R10 compared to 64% in P at 72 hrs. P cells treated with ibrutinib in combination with ricolinostat demonstrated synergy with RRR=0.46 (where RRR<1 connotes synergy) after 96 hour exposure to ibrutinib and ricolinostat. The combination led to modulation of HELIOS, p-BTK, p-AKT, SH3BP5, and FYN. These findings were confirmed in 2 additional ABC-DLBCL and MCL cell lines as well as in 2 primary patient samples of lymphoma: 1 CLL and 1 LPL with RRR of 0.54, and 0.52 respectively. In vivo confirmation of synergy is now underway in mouse models of Ly10 and R20-Ly10.The development of these ricolinostat drug resistant cell lines has proven to be a powerful tool to better understand the mechanistic role of HDAC6 in lymphoma and may potentially lead to the identification of biomarkers of response or resistance. In addition, we believe this strategy may offer a new way to identify rational and synergistic drug : drug combinations. Translation of these findings to the clinic are now under consideration. DisclosuresAmengual:Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. O'Connor:Seattle Genetics: Consultancy; Bristol-Myers Squibb Company: Consultancy; Mundipharma: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Acetylon Pharmaceuticals, INC: Consultancy.

Abstract B85: EZH2 plays a critical role in B-cell maturation and in non-Hodgkin's lymphoma: Interplay between EZH2 function and B-cell activation

Abstract The EZH2 inhibitor tazemetostat (EPZ-6438) is emerging as a promising therapeutic agent for the treatment of non-Hodgkin's Lymphoma (NHL). A significant body of work has now demonstrated in vitro and in vivo effects of EZH2 inhibition in preclinical models of lymphoma, in addition to objective clinical responses in early human trials. While EZH2 gain-of-function mutations clearly contribute to lymphomagenesis, patients with lymphomas harboring wild-type EZH2 also show responses to tazemetostat. This suggests a broad role of EZH2 in B-cell oncogenesis. Several recent mouse model studies have demonstrated the importance of wild-type EZH2 catalytic activity in the formation of germinal centers in non-diseased lymph nodes, suggesting a central role for EZH2 in B-lymphocyte maturation. These findings indicate that the importance of EZH2 to B-cell lymphoma likely lies in its ability to regulate B-cell differentiation. To understand the relationship between B-cell maturation and sensitivity to EZH2 inhibition, we evaluated changes in maturation markers and cellular proliferation following treatment of diffuse large B-cell lymphoma (DLBCL) cell lines with tazemetostat in combination with modulators of B-cell activation. Consistent with the importance of EZH2 in the regulation of B-cell differentiation, we observed increased expression of B-cell maturation markers in DLBCL cell lines treated with single agent tazemetostat in vitro. Furthermore, we demonstrate that tazemetostat pre-treatment of subsets of DLBCL cells lines (both EZH2 mutant and wild-type) can sensitize cells to inhibitors of B-cell activation pathways, which include glucocorticoids and BTK, MAPK and PI3K pathway inhibitors. Moreover, the anti-proliferative activity of single agent tazemetostat can be diminished or delayed by co-treatment with biological stimulators of B-cell activation including B-cell receptor ligation, CD40L, LPS and BAFF. Importantly, B-cell receptor ligation and co-stimulation agents have little proliferative effects on DLBCL cell lines on their own, suggesting that the protective function of these agents is directly related to the effects of EZH2 inhibition and not a generic stimulation of proliferation. Our findings suggest that EZH2 inhibition initiates a differentiation program that enables lymphoma cells to proceed through the normal processes of B-cell selection, growth regulation and maturation. Citation Format: Danielle Johnston, Dorothy Brach, Christopher Plescia, Alison Drew, Trupti Lingaraj, Natalie Warholic, Jesse J. Smith, Robert A. Copeland, Heike Keilhack, Elayne Penebre, Sarah K. Knutson, Scott Ribich, Michael J. Thomenius, Alejandra Raimondi. EZH2 plays a critical role in B-cell maturation and in non-Hodgkin's lymphoma: Interplay between EZH2 function and B-cell activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B85.

Dual Targeting of Protein Degradation Pathways with the Selective HDAC6 Inhibitor ACY-1215 and Bortezomib Is Synergistic in Lymphoma.

Pan-class histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. HDAC6 is a class IIb deacetylase that facilitates misfolded protein transport to the aggresome for degradation. We investigated the mechanism and therapeutic impact of the selective HDAC6 inhibitor ACY-1215 alone and in combination with bortezomib in preclinical models of lymphoma. Concentration-effect relationships were defined for ACY-1215 across 16 lymphoma cell lines and for synergy with bortezomib. Mechanism was interrogated by immunoblot and flow cytometry. An in vivo xenograft model of DLBCL was used to confirm in vitro findings. A collection of primary lymphoma samples were surveyed for markers of the unfolded protein response (UPR). Concentration-effect relationships defined maximal cytotoxicity at 48 hours with IC50 values ranging from 0.9 to 4.7 μmol/L. Strong synergy was observed in combination with bortezomib. Treatment with ACY-1215 led to inhibition of the aggresome evidenced by acetylated α-tubulin and accumulated polyubiquitinated proteins and upregulation of the UPR. All pharmacodynamic effects were enhanced with the addition of bortezomib. Findings were validated in vivo where mice treated with the combination demonstrated significant tumor growth delay and prolonged overall survival. Evaluation of a collection of primary lymphoma samples for markers of the UPR revealed increased HDAC6, GRP78, and XBP-1 expression as compared with reactive lymphoid tissue. These data are the first results to demonstrate that dual targeting of protein degradation pathways represents an innovative and rational approach for the treatment of lymphoma.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma.

Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell-specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development.