Granta-519 Cells Research Articles

Synthesis and Anti-Cancer Activity of the Novel Selective Glucocorticoid Receptor Agonists of the Phenylethanolamine Series.

Glucocorticoids (GCs) are widely used for treating hematological malignancies despite their multiple adverse effects. The biological response to GCs relies on glucocorticoid receptor (GR) transrepression (TR) that mediates the anticancer effects and transactivation (TA) associated with the side effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could offer greater benefits in cancer treatment. One of the well-characterized SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), exhibited anticancer activity; however, its translational potential is limited due to chemical instability. To overcome this limitation, we obtained CpdA derivatives, CpdA-01-CpdA-08, employing two synthetic strategies and studied their anti-tumor activity: 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol or CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. In lymphoma Granta and leukemia CEM cell lines, CpdA-03 ligand exhibited typical SEGRA properties, inducing GR TR without triggering GR TA. CpdA-03 effects on cell viability, growth, and apoptosis were similar to the reference GR ligand, dexamethasone (Dex), and the source compound CpdA. In vivo testing of CpdA-03 activity against lymphoma on the transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, in this work, we introduce a novel SEGRA CpdA-03 as a promising agent for lymphoma treatment with fewer side effects.

Inhibition of Autophagy Augments the Anticancer Activity of KPT-330 in Mantle Cell Lymphoma Cells

To investigate the influence of novel CRM1 inhibitor KPT-330 on the autophagy of mantle cell lymphoma (MCL) cells, and effect of KPT-330 on the prolifiration of MCL cells in the presence or absence of autophagy inhibitor. CCK-8 assay was used to detect the effect of KPT-330 on MCL cell lines Z-138， Jeko-1， Granta-519， Rec-1. The effect of KPT-330 on autophagy features were determined by detecting acidic vesicular organelles (AVO) by MDC staining under fluorescence microscope and detecting protein expression of LC3B-II assessed by Western blot. Further combined application of lysosomal inhibitor Chloroquine (CQ) was used to observe its effect on the increase of LC3B-Ⅱ caused by KPT-330. CalcuSyn 2.0 software was used to detected the Combination index (CI) of KPT-330 combined with CQ. The proliferation of MCL cell lines （Z-138, Jeko-1, Grant-519, Rec-1） could be inhibited by KPT-330 in a dose-dependent manner (r =0.930, 0.946, 0.691, 0.968 respectively). The number of acidic vesicular organelles (AVO) and the expression of LC3B-II were increased in KPT-330 treated Jeko-1 and Granta-519 cells in a dose-dependent manner (r Jeko-1=0.993, r Granta-519=0.971). LC3B-II protein amounts still increased upon KPT-330 treatment with the existence of lysosomal inhibitor CQ in Jeko-1 and Granta-519 cells, which was higher than CQ or KPT-330 single drug group. The combination of KPT-330 and CQ produced the synergistic effects on cells proliferation inhibition with CalcuSyn 2.0 analysis. KPT-330 can inhibit MCL cell proliferation and induce autophagy. KPT-330 combined with autophagy inhibitor CQ could produce synergistic anti MCL effects, providing experimental basis for clinical combination therapy.

Compatibility of Venetoclax and OPN51107 in Treatment of Mantle Cell Lymphoma

Introduction Mantle Cell Lymphoma (MCL) is the third most common form of B-Cell lymphoma and patients diagnosed tend to have poor long-term survival when compared to patients of other B-cell lymphoma subtypes. Deregulation of the apoptotic cellular machinery contributes to early acquirement of therapy resistance in MCL and contributes to the poor clinical outcomes observed in relapsed/refractory MCL patients. Developing therapeutic strategies using agents that favor a pro-apoptotic cellular environment are being investigated in pre-clinical and clinical studies. To this end, we evaluated the therapeutic effects of targeting the Bcl-2 related proteins using Venetoclax, a Bcl-2 inhibitor, and OPN51107, a BET inhibitor, in MCL pre-clinical models. OPN51107 targets BRD4 in B-cells, a protein involved in the preinitiation complex for transcription of miRNA inhibit the translation of BIM, a pro-apoptotic initiator in the Bcl-2 family. Methods Using a panel of MCL cell lines (HBL-2, Granta, Z-138, Mino, Jeko-1, and Rec-1) along with resistant cells of each cell line made to Cytarabine, we conducted pre-clinical targeting of Bcl-2 and BRD4 with Venetoclax and OPN51107 respectively. Single and double agent exposure experiments were performed at 72 hrs and coefficients of synergy were assessed using CalcuSyn. Synergy was determined from cellular viability using Cell Titer-Glo (Promega) and was read using a BioTek Synergy HTX multimode reader. During in vivo experiments, 6-week SCID mice were inoculated with 10 × 10 6 HBL-2 or Granta cells via tail vein injection. OPN51107 was administered at 5 and 20 mg/kg/dose PO Days (3-7, 10-14. And 17-21). Venetoclax was administered at 50 and 100 mg/kg/dose PO Days (3-7, 10-14, and 17-21). Bcl-2 family member protein expression was determined via western blot. Protein-protein interactions of members of the Bcl-2 family were also studied though Co-immunoprecipitation. Results In vitro exposure of MCL cells lines resulted in an up-regulation of BIM levels. Synergistic activity between Venetoclax and OPN51107 was observed in SCID mice bearing the Granta cells but not in animals inoculated with the HBL-2 cell line. The median survival for Granta bearing SCID mice treated with Venetoclax and OPN51107 was longer (26 days) when compared to animals treated with either Venetoclax (24 days) or OPN51107 (25 days) (P values: 0.000383 and 0.089 respectively). Ongoing studies are investigating protein to protein interactions between BIM and other Bcl-2 related proteins to further define the mechanisms responsible for the synergy observed between Venetoclax and OPN51107. Conclusion Previous in vitro activity presented a synergistic relationship on cell death from combination treatment of OPN51107 and Venetoclax. Our present research suggests that this relationship can also be replicated in vivo and further research has been done to help elucidate a possible mechanism of action responsible for the enhancement of activity seen. Continuing these directions to understand the synergy between OPN51107 and Venetoclax is necessary to advance therapies within MCL.

Combined Inhibition of Protein Kinase CSNK2 and BET Proteins As a Novel Therapeutic Strategy for Mantle Cell Lymphoma

Currently available therapeutic approaches for Mantle Cell Lymphoma (MCL), including the Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib, acalabrutinib and zanubrutinib, are not curative. Therefore, the discovery of novel molecules amenable of drug targeting is a research priority in the MCL field. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain (B) and extra terminal (ET) (BET) protein family that regulate chromatin modifications and assembly of transcriptional complexes. Inhibition of BET by available compounds have shown promising anticancer effect. In MCL, BET inhibitors, such as INCB054329 or JQ-1, have been shown to increase apoptosis through downregulation of the AKT-mTOR, ERK, and other B Cell Receptor (BCR)-triggered cascades. We and others have previously demonstrated that the S/T kinase CSNK2 supports chronic BCR activation in MCL by upregulating the AKT/PI3K and NF-κB pathways. Inactivation of CSNK2 results in efficacious induction of MCL cell death. Notably, BRD4 activity is regulated by CSNK2 and double inhibition of BRD4 and CSNK2 has shown significant activity in other tumor models. The most effective and tested CSNK2 chemical inhibitor is CX4945 (silmitasertib), but very recently a novel compound, SGC-CK2, has been developed. No data are available on the effects of SGC-CK2 on MCL cells. In this work, we have evaluated the effects of the combined inhibition of CSNK2 and BET proteins on MCL cell growth and survival signaling pathways. CSNK2 inhibitors employed were CX4945 and SGC-CK2. CSNK2 gene silencing was achieved with the generation of anti- CSNK2 short hairpin-RNA IPTG-inducible MCL cell clones. BET inhibitors used were JQ-1 and INCB054329. Cell survival, apoptosis and proliferation were investigated by AnnexinV/PI staining and FACS analysis, by Western blot (WB) detection of PARP cleavage and Mcl1 expression and by other cell proliferation assays. The synergic action of BET and CK2 inhibitors was assessed by the Chou-Talalay combination index method. CK2 expression and survival-related signaling components were analyzed by WB, qRT-PCR and protein expression assays. CX4945 and the novel inhibitor SGC-CK2 displayed a potent anti-tumor activity on MCL cells as judged by the effective induction of apoptosis and proliferation arrest. A synergistic effect of CSNK2 and BET inhibitors was observed in all the MCL cell lines tested, (Jeko-1, Rec-1, including Granta-519 that are known to be much less sensitive to ibrutinib) and with all the inhibitors tested. The same results were obtained in the CSNK2 gene silencing models. Preliminary data showed that the cooperative cytotoxic effect of CK2 inhibition and BET inhibition on MCL apoptosis was observed also in MCL patient derived B cells, but not in healthy B lymphocytes form buffy coat. CSNK2 inactivation was associated with strong down-regulation of c-Myc and Mcl-1 proteins and with hampered activation of NF-κB and PI3K/AKT-dependent signaling. Surprisingly, BET protein inhibition caused increased Mcl-1 protein expression and augmented Ser 529 p65-NF-kB phosphorylation in Granta-519 and Rec-1 cells. Remarkably, CK2 inactivation led to a robust reduction of the BET inhibitor-induced increase of Mcl1, and NF-kB Ser 529 phosphorylation, thus counteracting BET-inhibitors-evoked compensatory pathways that could favor apoptosis resistance. Therefore, combined CSNK2 and BET proteins inhibition could represent an innovative strategy for chemotherapy and BTKi-resistant MCL.

Inhibition of Lysine Acetyltransferases p300/CBP Overcomes BTK Inhibitor Resistance in Mantle Cell Lymphoma Cells

Background: Although Bruton's Tyrosine Kinase inhibitors (BTKis) are effective against relapsed refractory mantle cell lymphoma (MCL), the limited duration of therapeutic response and the emergence of resistance remain clinical issues. We conducted a CRISPR/Cas9 screen to identify the genes responsible for BTKi sensitivity and resistance. Materials and Methods: To identify the genes responsible for BTKi resistance, we used a CRISPR library (Kosuke Yusa, V3), targeting 18,740 human genes. We compared three BTKis, ibrutinib, acalabrutinib and tirabrutinib, using DMSO as a control. We transduced Granta519, an ibrutinib-resistant MCL cell line expressing Cas9 with the CRISPR library lentiviruses. Next, we counted sgRNA copy numbers using next generation sequencing after a two-week drug treatment. We identified 76 genes that sensitized cells to the BTKis upon knockout. After excluding common essential genes based on the Cancer Dependency Map portal (depmap.org/portal/), we ranked these genes according to the extent to which the combination of their knockout and BTKis inhibited cell proliferation. Results: Lysine acetyltransferases (KATs), p300 and CBP were ranked first and ninth, respectively. Knockout of p300 or CBP alone moderately decreased cell numbers, where as their combination with BTKi treatment significantly decreased cell numbers. To further validate this synergistic effect, we performed an MTS assay in combination with p300/CBP inhibitors (C-646 and A-485) and ibrutinib in MCL cell lines (Granta519 and Mino). The p300/CBP inhibitor increased ibrutinib sensitivity, leading to a up to four-fold greater suppression of cell viability. This effect was even greater than that of existing drugs, including venetoclax (a BCL-2 inhibitor), and idelalisib (a PI3K inhibitor), which are thought to have synergistic effects. The synergistic effect of a p300/CBP inhibitor and ibrutinib was also observed in long-term cultures for up to two weeks in both ibrutinib-resistant and ibrutinib-sensitive MCL cell lines. Furthermore, combination treatment resulted in an enhancement of G0/G1 cell cycle arrest and an increase in apoptotic cells in the MCL cell lines. To elucidate the mechanism underlying this synergistic effect, we measured the expression of IκBα mRNA as an indicator of NFκB activity by real-time PCR. In Granta519, ibrutinib alone caused an initial decrease in IκBα mRNA expression followed by a rebound increase. However, the addition of A-485 decreased the rebound and suppressed IκBα mRNA expression. These results indicate that ibrutinib and A-485 cooperatively suppress NFκB activity. The combination of ibrutinib and A-485 synergistically suppressed phosphorylation of PLCγ2 and of AKT as confirmed by Western blotting. This suggests that downstream PLCγ2 and AKT activation might be involved in ibrutinib resistance. Lentiviral overexpression of p300 in Granta519 cells enhanced phosphorylation of AKT. On the other hand, ibrutinib suppressed the expression of p300, and conversely, p300/CBP inhibitors suppressed the expression of BTK. These data suggest that the expression of p300 and BTK are dependent on each other. We hypothesize that direct or indirect interactions may occur through phosphorylation by BTK and acetylation by p300. Conclusion: Our CRISPR screen identified lysine acetyltransferases p300/CBP as BTK inhibitor resistance genes in human MCL cells. The combination of a p300/CBP inhibitor and a BTK inhibitor synergistically suppresses both BTK and AKT, leading to NFκB suppression. Taken together, we first identified that the combination of p300/CBP inhibition and BTK inhibition could be a promising strategy for treating patients with MCL.

Pre-Clinical Evaluation of a New-Generation Orally Bioavailable Dual Bcl-2/Bcl-Xl Inhibitor LP-118 in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is an aggressive form of non-Hopkins lymphoma with poor prognosis. The current frontline therapy of MCL is immunochemotherapy. For relapsed and refractory MCL, BTK inhibitor (BTKi) has become the standard of care, however, relapse is common after treatment with BTKi. Therefore, novel treatments with different mechanisms of action are needed for MCL patients. LP-118 is a new generation dual Bcl-2/Bcl-xL inhibitor with potent activity against wildtype and mutant Bcl-2 and moderate activity against Bcl-xL, and it is currently in Phase 1 clinical trials for multiple types of hematological cancers and solid tumors. In this study, we evaluated the preclinical activity of LP-118 in two MCL cell lines, REC-1 and Granta519, in vitro and in vivo. We also screened multiple combinations of LP-118 plus chemotherapeutic or targeted agents, in order to explore the optimal drug combinations for MCL treatment. Methods In vitro cell viability assay was performed with CellTiterGlo method in MCL cell lines REC-1 and Granta519 to examine cellular activity of LP-118. To determine the synergy of the combinations of LP-118 with standard of care agents, matrix combinations were performed with different concentrations of LP-118 and different drugs including vincristine, bendamustine, cytarabine, docetaxel, doxorubicin, cisplatin, and bortezomib. The data were analyzed using SynergyFinder (2.0), an online program for synergy scores. For in vivo experiments, Granta519 and REC-1-derived xenograft models were established and treated with different regimens: for Granta519 model, mice were treated with vehicle, BR (bendamustine, 25mg/kg, iv, day 1 and rituximab, 10mg/kg, iv, day 1), LP-118 (75mg/kg, po, qd, 28 days) in combination with BR. For REC-1 models, mice were treated with vehicle, LP-118 (50mg/kg, po, qd, 28 days) plus a BTK inhibitor LP-168 (25mg/kg, po, bid, 28 days), or ABT-199 (50mg/kg, po, qd, 28 days) plus ibrutinib (25mg/kg, po, qd,13 days, then bid, 28 days), and tumor growth/remission were examined to determine the in vivo anticancer activity. Results LP-118 is highly active in cell viability assays with Granta519 and REC-1 cells, with IC 50 of 0.6 nM and 1.96 nM, respectively. And compared to venetoclax, LP-118 is 8 to18 times more potent in REC-1 and Granta519 cells. In the matrix combination studies with REC-1 cells, combinations of LP-118 with SOC agents showed synergistic anticancer effects with all the tested drugs, including vincristine, bendamustine, cytarabine, docetaxel, doxorubicin, cisplatin, and bortezomib. Moreover, the combination of LP-118 with BR induced complete tumor regression of Grana519 xenografts in all mice (7/7) at day 45 with no significant body weight changes, whereas no complete tumor regression was observed in the BR group, indicating that combination of LP-118 with BR was more effective than the treatment with BR alone. Similarly, the combination of LP-118 with LP-168 (BTKi) also resulted in complete tumor regression in REC-1 xenograft models at day 40, while the combination of ABT-199 plus ibrutinib showed no significant tumor growth inhibition, suggesting that combination of LP-118 and LP-168, a new generation BTKi currently also in clinical trial, might be more effective against MCL than the combination of ABT-199 plus ibrutinib in clinic. Conclusion LP-118 showed encouraging anti-MCL activity as single agent and in combination with standard of care agents in vitro and in vivo, warranting further evaluation of these combinations in clinical trials.

Proinflammatory microenvironment promotes lymphoma progression in mice with high megakaryocyte and TPO levels.

Platelets have been shown to enhance the survival of lymphoma cell lines. However, it remains unclear whether they play a role in lymphoma. Here, we investigated the potential role of platelets and/or megakaryocytes in the progression of Eμ-myc lymphoma. Eμ-myc tumor cells were transplanted into recipient wild-type (WT) control, Mpl-/-, or TpoTg mice, which exhibited normal, low, and high platelet and megakaryocyte counts, respectively. TpoTg mice that underwent transplantation exhibited enhanced lymphoma progression with increased white blood cell (WBC) counts, spleen and lymph node weights, and enhanced liver infiltration when compared with WT mice. Conversely, tumor-bearing Mpl-/- mice had reduced WBC counts, lymph node weights, and less liver infiltration than WT mice. Using an Mpl-deficient thrombocytopenic immunocompromised mouse model, our results were confirmed using the human non-Hodgkin lymphoma GRANTA cell line. Although we found that platelets and platelet-released molecules supported Eμ-myc tumor cell survival invitro, pharmacological inhibition of platelet function or anticoagulation in WT mice transplanted with Eμ-myc did not improve disease outcome. Furthermore, transient platelet depletion or sustained Bcl-xL-dependent thrombocytopenia did not alter lymphoma progression. Cytokine analysis of the bone marrow fluid microenvironment revealed increased levels of the proinflammatory molecule interleukin 1 in TpoTg mice, whereas these levels were lower in Mpl-/- mice. Moreover, RNA sequencing of blood-resident Eμ-myc lymphoma cells from TpoTg and WT mice after tumor transplantation revealed the upregulation of hallmark gene sets associated with an inflammatory response in TpoTg mice. We propose that the proinflammatory microenvironment in TpoTg mice promotes lymphoma progression.

IKS03, a Next Generation CD19-Targeted Antibody Drug Conjugate, Shows Potent Activity in Preclinical Models of Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a common subtype of non-Hodgkin lymphoma (NHL). Although many patients respond to frontline chemoimmunotherapy, and treatment options for patients with relapsed or refractory DLBCL have improved with the success of CAR T cell therapies, clinical outcome for many patients is still suboptimal. CD19 is widely expressed in DLBCL tumor cells, with normal tissue expression limited to B-cells, making it an attractive target for targeted therapies including antibody drug conjugates (ADC). IKS03 is a CD19-targeting ADC with a PBD dimer pro-drug payload that induces DNA crosslinking and blocks DNA replication ultimately leading to cell death. Following tumor selective binding and uptake, IKS03 requires intracellular lysosomal processing of beta-glucuronidase protecting groups to fully activate the payload which minimizes systemic release of the PBD dimer in human plasma. IKS03 is derived from a human antibody that binds with nanomolar affinity to human and cynomolgus monkey CD19. It is generated by site-specific bioconjugation yielding a homogeneous conjugate with a drug to antibody ratio of 2. In vitro, IKS03 induces specific and dose dependent cell killing of diverse CD19+ lymphoma cell lines with sub-nanomolar EC50 values. Conversely, in vitro cytotoxicity of the conjugate against normal human primary peripheral blood mononuclear cells was attenuated with an EC50 of approximately 1 μM, similar to a non-PBMC binding ADC bearing the same PBD pro-drug. The unconjugated payload more potently reduced PBMC viability with a sub-nanomolar EC50. This is consistent with low non-specific payload release from the conjugate underscoring the improved properties of the PBD pro-drug design. Pharmacokinetics of IKS03 was assessed in a repeat dose toxicity study in monkeys and the plasma concentrations of total ADC and total antibody were determined by LC-MS/MS across the 3-week dosing interval. Overall mean half-life for total ADC was approximately 8 days and clearance rate was slow (mean 0.2 mL/h/kg). Total ADC peak concentration, total plasma exposure and half-life were comparable to that of total antibody demonstrating favorable ADC stability in plasma with limited systemic payload release. In vivo efficacy was evaluated in CD19-expressing preclinical human xenograft models representing different subtypes of B-NHL. Granta-519 cells contain the CCND1 t(11;14) translocation often present in mantle cell lymphoma (MCL). IKS03 was highly active against Granta-519 xenografts in SCID mice with complete tumor regressions observed with a single dose of 0.1 mg/kg. Efficacy was further evaluated in CD19-positive, low passage DLBCL patient-derived xenograft (PDX) models of known genomic profiles. IKS03 was highly active yielding complete tumor regressions following a single dose of 0.3 mg/kg in a refractory lymphoma Bcl2+ PDX model derived from a patient with no response to prior CHOP treatment. Post tumor collection, this patient was also refractory to EPOCH chemotherapy. A second xenograft model was derived from a treatment naïve high-grade 'triple hit' lymphoma patient (BCL2+; BCL6+; MYC+; XP01 amplified). Post tumor collection, this patient was refractory to R-CHOP therapy with disease progression within 2 months. Tumors rapidly progressed in this model, yet single dose treatment with IKS03 resulted in significant tumor growth inhibition at 0.3 mg/kg and complete tumor regressions at 1 mg/kg. Likewise, in a pre-treated MYC-amplified GCB lymphoma model, a single IKS03 dose of 0.3 mg/kg inhibited tumor growth and 1 mg/kg yielded complete inhibition of tumor growth. In conclusion, IKS03 is highly effective in preclinical MCL and DLBCL xenograft models that contain some of the genetic alterations commonly found in relapsed/refractory NHL patients in need of treatment. Preclinical data further demonstrates that IKS03's advanced ADC design results in specific target-dependent in vitro activity, excellent plasma stability and allows for effective targeting of B-cell tumors while sparing normal CD19-negative cells.

Regulation of TRIP13 on Proliferation and Apoptosis of B-Cell Lymphoma Cells and Its Mechanism

To explore the regulatory effect of TRIP13 on the proliferation and apoptosis of B-cell lymphoma cells and its possible molecular mechanism by knocking down/overexpressing TRIP13 on the cell lines Granta-519 and JVM-2. Lentiviral transfection technology was used to construct Granta-519 and JVM-2 cells with knocked down or overexpressed TRIP13 and their control cells. The efficiency of transfection was determined by fluorescence microscopy. The efficiency of knockdown and overexpression was evaluated by real-time quantitative PCR and Western blot. The proliferation was detected by CCK-8 assay. The apoptosis was detected by the Annexin V-APC single staining. The cell cycle was detected by the PI staining. The expression levels of P53, MDM4, and BCL-2 were evaluated by Western blot. After TRIP13 was knocked down, the proliferation ability of Granta-519 and JVM-2 cells was significantly reduced, and the apoptosis rate significantly increased. After TRIP13 was overexpressed, the proliferation ability of Granta-519 and JVM-2 cells was significantly enhanced, and the apoptosis was significantly reduced. After TRIP13 was knocked down, Granta-519 cells had obvious G1 phase arrest, and JVM-2 cells had obvious G1 and G2/M phase arrest. After TRIP13 was knocked down in Granta-519 cells, the expression of BCL-2 protein decreased, while MDM4 protein increased. After TRIP13 was overexpressed, the expression of MDM4 protein decreased. After TRIP13 was overexpressed in JVM-2 cells, the expression of BCL-2 protein increased. TRIP13 promotes the proliferation of B-cell lymphoma cells, inhibits their apoptosis, and affects their proliferation and apoptosis by participating in the regulation of the cell cycle. TRIP13 promotes the expression of BCL-2 proteins and inhibits the expression of MDM4 protein in B-cell lymphoma cells.

Momelotinib and Citarinostat: Co-Targeting JAK2/STAT3 and HDAC6 in Lymphoid Malignancies, a New Potential Therapeutic Combination

Background . The JAK2/STAT3 signaling pathway is abnormally activated in hematological malignancies. HDAC6 has been reported to be overexpressed in primary and cultured multiple myeloma cells and B and T-cell lymphoma. Momelotinib (CYT-387) is a potent inhibitor of JAK1 and JAK2 that demonstrated efficacy in patients with primary and secondary myelofibrosis. Citarinostat (ACY-241), a second generation HDAC6 selective inhibitor, has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drugs candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. Both drugs are currently under investigation in clinical trials, they show a good toxicity profile and are both orally available.Methods. Screening: momelotinib and citarinostat alone and in combination were tested in 12 lymphoid malignant cell lines: B-cell lymphomas (WSU-NHL, RL, Karpas-422), mantle cell lymphoma (Granta-519, Jeko-1), cutaneous T cell lymphoma (Hut-78), anaplastic large cell lymphoma (Karpas-299), Hodgkin lymphoma (L-1236, L-540), multiple myeloma (U266, RPMI8266) and chronic lymphocytic leukemia (MEC-1). IC50 values of each drug were calculated based on momelotinib concentrations (1 - 10 μM), and citarinostat (1- 100 μM) after 24 - 48 h. Serial dilutions of the two agents were assessed using low concentrations and the interaction was evaluated using the Chou-Talalay method. Cell viability was measured by MTT assay. Cell cycle was determined by flow cytometry. Cell apoptosis was detected by Annexin V/PI staining and apoptosis-related proteins (Bcl2, Bcl-xL, Bax, Bim, PARP and caspase 8) were detected by western blotting. Enzymatic activity of caspases 3, -9 was measured using colorimetric assay. JAK2/STAT3 and the related proteins were analysis by western blotting.Results . Momelotinib alone exhibited anti-proliferation potency in the cell lines examined with IC50 values ranging from 1.8 to 9.6 μM. Exposure for 24 - 48 h with citarinostat alone resulted in time- and dose-dependent inhibition of cell growth with IC50 values ranging from 1.51 to 60 μM. After 24 h, low concentrations of the two drugs, momelotinib (1 μM) and citarinostat (4 μM) had a synergistic effect in WSU-NHL, RL, Karpas-422, Jeko-1, Hut-78, Karpas-299, L-540, RPMI8226 and U266 cells with CI (combination index) values < 1 and antagonist effect in L-1236, Granta-519 and Mec-1 cells with CI > 1. From the panel, we selected three lymphoid cell lines (WSU-NHL, RL, L-540) which were particularly sensitive to the drug combination and two cell lines (L-1236, Granta-519) that showed an IC > 1. While in these cell lines the combination treatment had minimal or no cytotoxic effect, WSU-NHL, RL and L-540 cells showed an increase of cells in sub-G0/G1 phase and a reduction in S phase after 24 h. Compared with single agents, the percentage of apoptotic cells ranged from 35% to 42%. Apoptosis induced by the drug combination was exerted mainly via the mitochondrial apoptotic pathway (intrinsic apoptotic pathway) as demonstrated by upregulation of caspase -9 that was especially evident in WSU-NHL and L-540 with a fold induction of 3.2 and 3.3 compared to control. The apoptosis was associated with activation of caspase-3 and PARP which were prevented by the ZVAD-fmk broad caspase inhibitor. No caspase-8 activation (extrinsic apoptotic pathway) was induced within 24 h of combination treatment. The effect was mediated by the increased expression of the pro-apoptotic proteins Bax and Bim and downregulation of key anti-apoptotic regulators, Bcl2 and Bcl-xL. In addition, momelotinib/citarinostat combination inhibited the phosphorylation of JAK2 and its downstream mediators, including STAT3 and STAT5.Conclusions. These preliminary results indicate that momelotinib can be potentially combined with the HDAC6 inhibitor citarinostat in lymphoid malignancies. Due to the good toxicity profile and the oral administration the combination has the potential for a completely orally therapeutic approach. The study is still ongoing and further investigation is required to better define the underlying molecular mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI &amp;lt; 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI &amp;gt; 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Oncogenic MALT1 Promotes Cell Survival and Mediates Ibrutinib Resistance and Ibrutinib-Venetoclax Resistance in Mantle Cell Lymphoma

Both as monotherapies and in combination, the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax have proven to be efficacious and are now widely used treatment options for mantle cell lymphoma (MCL) patients. However, mono- and dual- resistance frequently develops, necessitating investigation into the mechanisms mediating resistance to these therapies. To investigate the mechanism of ibrutinib resistance, we generated two ibrutinib-resistant cells due to marked BTK knockdown via CRISPR/CAS9 from JeKo-1, which is ibrutinib-sensitive and venetoclax-resistant. To understand the mechanism of venetoclax resistance, we generated three venetoclax-resistant cell lines with acquired resistance via chronic exposure to increasing doses of venetoclax from ibrutinib/venetoclax double sensitive Mino and Rec-1 cells, and ibrutinib-resistant but venetoclax-sensitive Granta519 cells. All these paired cell lines with various resistance to ibrutinib and venetoclax were subject to whole transcriptome sequencing of these paired MCL cell lines. We discovered that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is significantly overexpressed in ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells, especially in cells with BTK knockdown. This was further validated in primary MCL patient cells (n=24). Interestingly, MALT1 overexpression inversely correlates with CARD11 expression and enhances non-canonical NF-κB signaling, suggesting a switch from a highly-dependent BTK-CARD11 mechanism to an independent mechanism in both ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells. Chromosomal translocations of MALT1 are the hallmarks of MALT lymphoma, which result in oncogenic fusion of MALT1 products. MALT1 is constitutively active in driving aggressive ABC-type DLBCL. This indicates MALT1 can be oncogenic when its activity is dysregulated. Therefore, we hypothesized that constitutive MALT1 activity may be responsible for the resistance to ibrutinib and venetoclax in MCL cells. To demonstrate this relationship, MALT1 ablation using genetic manipulation resulted in significant growth defects both in vitro and in vivo. Pharmaceutical approaches using MALT1 inhibitor MI-2 resulted in similar effects on cell survival and using cell viability assays. Whole transcriptome sequencing analysis revealed that MYC, NF-kB, ROS, cell cycle and mTOR signaling are the most significantly downregulated pathways upon MI-2 treatment. Intriguingly, MYC, NF-kB, PI3K-AKT-mTOR and mTOR signaling pathways were reported to be upregulated in ibrutinib-resistant MCL cells compared to sensitive MCL cells. To address this further, proteomics analysis by reverse phase protein array (RPPA) using more than 400 antibodies confirmed that MI-2 significantly downregulated AKT-mTOR signaling. NF-kB modulation, ROS production, AKT-mTOR, and metabolism changes were further confirmed through multiple biochemical approaches. In addition, MI-2 treatment resulted in a dramatic reduction of MALT1 expression, suggesting that MI-2 treatment affected both its scaffold and paracaspase activities. Furthermore, MI-2 treatment resulted in significant inhibition of in vivo tumor growth of ibrutinib-venetoclax dual-resistant MCL subcutaneous xenografts and tumor homing to the spleen and bone marrow in an ibrutinib-venetoclax dual-resistant MCL patient-derived xenograft (PDX) mouse model. In conclusion, we discovered that MALT1, an essential regulator of NF-κB signaling, is hyperactive in ibrutinib-resistant cells and ibrutinib-venetoclax dual-resistant MCL cells, which puts MALT1 forward as a potentially new therapeutic target in ibrutinib and venetoclax-resistant MCL tumors. Genetic depletion or pharmaceutical inhibition of MALT1 resulted in remarkable defects in cell survival and cell proliferation. The MALT1 inhibitor MI-2 proved its in vivo potency by its pro-apoptotic effect and its significant tumor growth inhibition. In conclusion, targeting a hyperactive MALT1 is a promising therapeutic strategy that could lead to clinical implementation of a new treatment strategy meant to overcome ibrutinib and ibrutinib-venetoclax dual resistance in MCL patients by reversing the NF-kB and ROS/mTOR- mediated resistance in these tumors. Disclosures Wang: Targeted Oncology: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; MoreHealth: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses.

TOSO interacts with SYK and enhances BCR pathway activation in chronic lymphocytic leukemia.

BackgroundTOSO, also named Fas inhibitory molecule 3 (FAIM3), has recently been identified as an immunoglobulin M (IgM) Fc receptor (FcμR). Previous studies have shown that TOSO is specifically over-expressed in chronic lymphocytic leukemia (CLL). However, the functions of TOSO in CLL remain unknown. The B-cell receptor (BCR) signaling pathway has been reported to be constitutively activated in CLL. Here, we aimed to investigate the functions of TOSO in the BCR signaling pathway and the pathogenesis of CLL.MethodsWe over-expressed TOSO in B-cell lymphoma cell lines (Granta-519 and Z138) by lentiviral transduction and knocked down TOSO by siRNA in primary CLL cells. The over-expression and knockdown of TOSO were confirmed at the RNA level by polymerase chain reaction and protein level by Western blotting. Co-immunoprecipitation with TOSO antibody followed by liquid chromatography coupled with tandem mass spectrometry (IP/LCMS) was used to identify TOSO interacting proteins. Western blotting was performed to detect the activation status of BCR signaling pathways as well as B-cell lymphoma 2 (BCL-2). Flow cytometry was used to examine the apoptosis of TOSO-over-expressing B lymphoma cell lines and TOSO-down-regulated CLL cells via the staining of Annexin V and 7-AAD. One-way analyses of variance were used for intergroup comparisons, while independent samples t tests were used for two-sample comparisons.ResultsFrom IP/LCMS, we identified spleen tyrosine kinase (SYK) as a crucial candidate of TOSO-interacting protein and confirmed it by co-immunoprecipitation. After stimulation with anti-IgM, TOSO over-expression increased the phosphorylation of SYK, and subsequently activated the BCR signaling pathway, which could be reversed by a SYK inhibitor. TOSO knockdown in primary CLL cells resulted in reduced SYK phosphorylation as well as attenuated BCR signaling pathway. The apoptosis rates of the Granta-519 and Z138 cells expressing TOSO were (8.46 ± 2.90)% and (4.20 ± 1.21)%, respectively, significantly lower than the rates of the control groups, which were (25.20 ± 4.60)% and (19.72 ± 1.10)%, respectively (P < 0.05 for both). The apoptosis rate was reduced after knocking down TOSO in the primary CLL cells. In addition, we also found that TOSO down-regulation in primary cells from CLL patients led to decreased expression of BCL-2 as well as lower apoptosis, and vice versa in the cell line.ConclusionsTOSO might be involved in the pathogenesis of CLL by interacting with SYK, enhancing the BCR signaling pathway, and inducing apoptosis resistance.

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in Mantle Cell Lymphoma Models

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) that accounts for ~7% of all NHL in the U.S. MCL is associated with aberrant activation of the Wnt signaling pathway, which plays a key role in the survival and maintenance of MCL-initiating cells. Many MCL patients experience relapse and subsequent disease progression due to chemoresistance following initial therapy; hence, novel therapies are needed. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. The purpose of these studies was to examine the antitumor activity of SM09419 in preclinical models of MCL. SM09419 potently inhibited both CLK1-CLK4 (IC50 for all &lt;0.02 µM) and Wnt signaling pathway (average EC50=0.068 µM) activities. In REC-1 and GRANTA-519 MCL cells, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt-related genes (CCND1, LEF1, TCF7) and proteins vs. vehicle. In tests on 5 MCL cell lines, cell proliferation was strongly impaired by SM09419 across all lines (average EC50=0.102 µM [0.021-0.236]). SM09419 also induced apoptosis in REC-1 and GRANTA-519 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression vs. vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD 20-21 days) were assessed in mice bearing REC-1 and JeKo-1 flank xenografts (n=5/group). In REC-1 xenografts, strong tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with 12.5, 25, and 50 mg/kg SM09419 (TGI 88% [p&lt;0.01], 100%, and 100% [p&lt;0.001], respectively), and the two highest doses induced complete tumor regression in all mice from D14. Similarly, in JeKo-1 xenografts, SM09419 (12.5 and 25 mg/kg) induced significant TGI vs. vehicle (71% and 100%, respectively; p&lt;0.0001) with complete tumor regression at 25 mg/kg, whereas acalabrutinib (50 mg/kg BID) was not efficacious (27% TGI) when tested in parallel. SM09419 25mg/kg induced reversible suppression of phospho-SRSF6 protein and inhibited Wnt pathway-related gene expression (TCF7 and DVL2) in JeKo-1 tumors in a single-dose PD study, demonstrating downstream target engagement in vivo. SM09419 was also assessed in 2 patient-derived xenograft (PDX) mouse models of MCL. PDX cells were injected intravenously and treatment was initiated upon 8-12% engraftment of human CD45+CD19+ cells in peripheral blood. In the first model, derived from a patient who was progressive after 8 modalities including ibrutinib, SM09419 (25 mg/kg QD) increased survival vs. vehicle (100% through D26 vs. 0% by D12, respectively; n=6/group) and suppressed MCL engraftment in the blood (12% at D26 vs. 69% at D8 and D12, respectively; p=0.002) and bone marrow (30% at D26 vs. 91% at D8 and D12, respectively; p=0.002). In the second model, derived from a patient refractory after ibrutinib and anti-PDL1 treatment, SM09419 (25 mg/kg QD) significantly suppressed MCL engraftment vs. vehicle in the blood (8% vs. 72%), bone marrow (20% vs. 57%), and spleen (15% vs. 96%) at D28 (study end; p&lt;0.001 for all; n=4/group). In addition, SM09419 greatly inhibited splenomegaly vs. vehicle (0.04 g vs. 0.4 g, respectively; p&lt;0.001). In a subsequent experiment in the same model, mice (n=7/group) were treated with 12.5 or 25 mg/kg SM09419 or vehicle for 12 weeks (to D85). Blood MCL engraftment at D41 was significantly lower in mice treated with SM09419 (40% at 12.5 mg/kg and 23% at 25 mg/kg) vs. vehicle (88%; p&lt;0.01 and p&lt;0.001, respectively). SM09419 dose-dependently increased survival (28.6% at 12.5 mg/kg and 85.7% at 25 mg/kg at D85) vs. vehicle (0% at D63); survival was maintained in both dose groups during post-treatment monitoring (to D99). SM09419 was well tolerated in all tested mouse models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation Wnt signaling pathway activity, and cell proliferation and induced apoptosis in MCL cell lines. The strong in vivo antitumor effects observed as a single agent suggest that SM09419 may provide a clinical benefit for patients with treatment-resistant or refractory MCL. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

Improvement of the rituximab-induced cell death by potentiation of the store-operated calcium entry in mantle cell lymphoma cell lines.

Mantle Cell Lymphoma (MCL) is one of the worst lymphomas with a median overall survival of 3 to 4 years. Even if the use of rituximab was a great step in therapy, patients commonly develop resistance and relapse. New therapies or complement of existing therapies should be developed. Using spectrofluorimetry, we found that the resting cytosolic Ca2+ ion concentration [Ca2+]cyt of MCL patients cells and MCL cell lines was increased. This increase is correlated with a larger store-operated calcium entry (SOCE) amplitude which is responsible for the Ca2+ ions influx. Furthermore, using a SOCE potentiating agent, we demonstrated that in the MCL Rec-1 cell line, the SOCE is already activated in resting conditions. Interestingly, this potentiating agent alone, by disturbing the SOCE, induced the apoptosis of Rec-1 cells with the same efficacy than rituximab. The use of the potentiating agent in addition to rituximab strengthens the rituximab-induced apoptosis of rituximab-sensitive Granta-519 and Rec-1 cells. However, this potentiating agent cannot convert the Jeko-1 rituximab-resistant to a rituximab-sensitive cell line. Our results confirm that the use of compound acting on the Ca2+ homeostasis could be a new target of interest in complement to existing therapies.

The lipid raft protein NTAL participates in AKT signaling in mantle cell lymphoma

Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold (n = 8) and decreased tumor weight by 2.7-fold (n = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.

Oncogenic Role of Chromatin Modifier Polycomb Repressive Complex-2 in Mantle Cell Lymphoma

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. DisclosuresNo relevant conflicts of interest to declare.

Combination of Momelotinib and Citarinostat Show Strong Synergistic Effect in Lymphoid Cell Lines Co-Targeting JAK2/STAT3 and HDAC6

▪Introduction. Janus kinases (JAKs) are well described signaling kinases comprising four family members JAK1, JAK2, JAK3 and TYK2 that are essential in hematological malignancy, as JAK mutations have been shown to contribute to the pathogenesis of myeloproliferative disorders. Momelotinib is a potent inhibitor of JAK1/JAK2 that demonstrated efficacy in patients with primary and secondary myelofibrosis. HDAC6 has been reported to be overexpressed in lymphoid cells and its inhibition has demonstrated activity in preclinical and clinical study of lymphoproliferative disease. Citarinostat, a second generation HDAC6 selective inhibitor, is a well-tolerated compound compared with nonselective HDAC inhibitors, with reduced potency against Class I HDACs while retaining anticancer effectiveness. Both drugs are currently under investigation in clinical trials, they show a good toxicity profile and are both orally available.Methods. Momelotinib and citarinostat alone and in combination were tested in 12 lymphoid cell lines: WSU-NHL, RL, Karpas422 (follicular lymphoma), Granta519, Jeko1 (mantle cell lymphoma), Hut78 (cutaneous T cell lymphoma), Karpas299 (anaplastic large cell lymphoma), L540, L1236 (Hodgkin's lymphoma), U266, RPMI8266 (multiple myeloma) and MEC1 (chronic lymphocytic leukemia).Synergistic interaction was evaluated using the Chou-Talalay method. Annexin V staining, ROS generation, cell cycle and migration assay were determined by flow cytometry. ATP levels and Mitochondrial Membrane Potential (ΔΨm) were evaluated by fluorometric assay. Lactate levels and Cyt-C were evaluated by colorimetric assay. Activity of caspases-8,-9 and 3 was measured using colorimetric assay. ER stress and apoptosis-related proteins and JAK2/STAT3 were detected by western blotting. Clonogenic survival was studied with the methylcellulose clonogenic assay. Co-cultures with bone marrow stromal cells were also performed.Results. The combination of momelotinib (1 μM) and citarinostat (4 μM) at 24 h showed a synergistic effect in WSU-NHL, RL, Karpas422, Jeko1, Hut78, Karpas299, L540, RPMI8226 and U266 cells with CI values < 1 and antagonist effect in L1236, Granta519 and Mec1 cells with CI > 1. We studied seven lymphoid cell lines (WSU-NHL, RL, Karpas422, Jeko1, L-540) which were particularly sensitive to the drug combination and two cell lines (L-1236, Granta-519) that showed an IC > 1. Drug combination exhibited a strong cytotoxicity, evidenced by reduction of mitochondrial depolarization, depletion of ATP and lactate levels and Cyt-C release from the mitochondria but also by increase in cellular apoptotic index and reactive oxygen species levels, leading to arrest in the sub-G0/G1 phase of the cell cycle. Apoptosis induced by the drug combination was exerted via the mitochondrial apoptotic pathway as demonstrated by upregulation of caspase-9 that was especially evident in WSU-NHL and Karpas422 with a fold increase of 3.2 and 3.8. The extrinsic apoptotic pathway was active in Karpas422 and Jeko1 cells as evaluated by upregulation of caspase-8 but not in WSU-NHL, RL and L540. The apoptosis was associated with activation of caspase-3, PARP and with increased hallmarks of ER stress and was mediated by the increased expression of the pro-apoptotic proteins Bad, Bax and Bim and downregulation of Bcl2, Bcl-xL and Mcl-1. Drug combination inhibited the migration induced by CXCL12 (chemoattractant known as stromal-cell derived factor-1, SDF-1α), reduced clonogenic survival and suppressed cell viability of lymphoid cells when co-cultured with bone marrow mesenchymal stromal cells targeting JAK2/STAT3 pathway and confirming acetylation of acetyl-α tubulin.Conclusion. Due to the good toxicity profile and the oral administration, combined therapy with momelotinib and citarinostat may represent a promising novel therapeutic modality for hematological malignancies. The study is ongoing and further investigation is required. DisclosuresNo relevant conflicts of interest to declare.

UBR5 Mutations in Mantle Cell Lymphoma Lead to Increased Proliferation through a Cyclin D1-Dependent Mechanism

Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma, with patient outcomes inferior to most other lymphoma subtypes. Recent progress in describing recurrent somatic gene mutations has led to a better understanding of MCL pathogenesis. However, the functional and clinical implications of many alterations remain to be elucidated. Here, to uncover the role of recurrent UBR5 gene mutations in lymphomagenesis, we studied a cohort of 248 MCL patients by targeted sequencing and performed genome editing of MCL-derived cell lines to investigate UBR5-mutation associated phenotypes in vitro. We identified deleterious UBR5 exon 58 hotspot mutations in 8% of MCL patients, all of which were mutually exclusive with CCND1 mutations. Proteomics analysis of Granta-519 and Jeko-1 cell lines with engineered UBR5 exon 58 indel mutations showed differential expression of genes involved in cell cycle and ubiquitination, and led to the discovery of decreased phosphorylation of CCND1 in the UBR5-mutated lines. Accordingly, in vitro studies of engineered genome-edited Granta-519, Jeko-1 and Mino cells revealed accumulation of cells in the S phase of the cell cycle, increased phosphorylation of retinoblastoma protein (Rb), and increased lymphoma cell proliferation. Our results demonstrate that UBR5 mutations, in addition to the hallmark t(11;14) translocation drive proliferation of MCL cells, potentially rendering mutation-carrying cells more sensitive to targeted therapies. DisclosuresGascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Roche: Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Tioma: Research Funding.

SAMHD1 Is Variably Expressed in Mantle Cell Lymphoma and Correlated to SOX11 but Not to Survival

IntroductionMantle cell lymphoma (MCL) is an incurable disease with a median survival of 3-5 years. Most cases express SOX11, a transcription factor associated with MCL pathobiology, which serves as a diagnostic marker. Current standard first-line therapy for younger MCL patients is rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and cytarabine (Ara-C), consolidated with high dose chemotherapy with autologous stem cell transplantation (ASCT), which is associated with prolonged survival (Eskelund et al. Br J Haematol 2016). Ara-C metabolizes into its active triphosphate form Ara-CTP which inhibits proliferation of the malignant cells. Recent reports suggest that the expression of the mammalian dNTPs hydrolase SAMHD1 determines response to Ara-C in acute myeloid leukemia (AML) by hydrolyzing Ara-CTP and thus by diminishing the anti-proliferative properties of Ara-C. Consequently, in vitro downregulation of SAMHD1 resulted in sensitization of AML cells to Ara-C (Herold et al., Nat Med 2017). SAMHD1 also exhibits anti-tumor properties via regulation of dNTP pool and is recurrently mutated in CLL (Clifford et al. Blood 2014) and T-PLL (Johansson et al. Blood Cancer J. 2018). In CLL a role in DNA-repair has been suggested (Clifford et al. Blood 2014). Thus, SAMHD1 may function as a tumor suppressor. In this study, we investigated for the first time the expression patterns of SAMHD1 in MCL and its association to clinical outcome, especially in patients receiving Ara-C as part of induction for ASCT.MethodsSAMHD1 and SOX11 expression was investigated by qPCR, WB and IHC on whole tissue sections or tissue microarrays. MCL cell lines were treated with gene-specific siRNA for SAMHD1 or SOX11. Data on overall survival (OS) was retrieved from patient records. For patients included in the Nordic MCL2 and MCL3 trials data on progression free survival (PFS) and OS were retrieved.ResultsInitial IHC showed that expression of SAMHD1 is high in normal T-cells and macrophages and low in cells in the mantle zones of reactive tonsils. Co-staining with CD20 in a heterogeneously treated cohort of primary MCL (n=104) showed a large variation between cases (median 73.15%, range: 0.4%-99.6%) and the staining intensity was lower than in T-cells. Sixty two/104 samples were also evaluated for SOX11 expression and the percentage of SOX11 positive cells moderately correlated with SAMHD1 expression (Spearman correlation coefficient 0.27, p=0.036). Analysis of mRNA expression (normalized to mRNA levels of respective genes in Granta519 cell line) of SAMHD1 (median RFI: 2.09, range: 0.18-10.69) and SOX11 (median RFI: 2.00, range: 0.00-7.11) in flow cytometry sorted primary MCL cells (n=19) showed a trend for correlation (Spearman correlation coefficient 0.45, p=0.053). However, downregulation of SOX11 by siRNA did not alter the expression of SAMHD1 and neither did downregulation of SAMHD1 by siRNA change the expression of SOX11 suggesting that the observed correlation is not due to a mutual regulation. We investigated the relation of SAMHD1 expression to clinical and pathological parameters in this cohort and found no correlation to OS, blastoid morphology, proliferation (% Ki67+ tumor cells) nor p53 positivity (>20% positively stained cells). Further, a cohort of patients treated within the Nordic MCL2 and MCL3 protocols (n=51) were investigated for SAMHD1 expression by IHC. Interestingly the distribution of SAMHD1 expression was different in this cohort (median 34.9%, range: 2.2%-97.1%; Mann-Whitney p=0.0019). Survival analysis showed a trend for better PFS and OS in patients with high (>75% SAMHD1 positive cells) and low (<25% positive cells) SAMHD1, although the differences were not significant (Kaplan-Meier with log rank test, p=0.053).ConclusionsIn MCL the expression of SAMHD1 varies over a broad range and correlates to expression of SOX11. However, no significant difference in PFS or OS among patients receiving Ara-C containing induction chemotherapy is found in this study. DisclosuresNo relevant conflicts of interest to declare.