PRMT5 INHIBITION RESTARTS A PRO‐APOPTOTIC PROGRAM AND CREATES VULNERABILITY TO COMBINATION TREATMENT WITH BCL‐2 INHIBITOR VENETOCLAX IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is an incurable B cell malignancy, defined by the t(11;14) translocation and comprises 3-6% of non-Hodgkin lymphomas diagnosed annually. MCL is associated with a poor prognosis due to emergence of resistance to immuno-chemotherapy and targeted agents. Due to the late median age of diagnosis, aggressive chemotherapy and stem cell transplantation are often not realistic options. The average overall survival of patients with MCL is 5 years and for the majority of patients who progress on targeted agents like ibrutinib, survival remains at a dismal 3-8 months. There is a major unmet need to identify new therapeutic approaches that are well tolerated by elderly patients to improve treatment outcomes and quality of life. Our group has identified the type II protein arginine methyltransferase enzyme, PRMT5, to be dysregulated in MCL and to promote growth and survival by supporting the cell cycle, PRC2 activity, and signaling via the BCR and PI3K/AKT pathways. We have developed first-in-class selective inhibitors of PRMT5 and, in collaboration with Prelude Therapeutics, we have demonstrated that novel SAM-competitive PRMT5 inhibitors provide potent anti-tumor activity in aggressive preclinical models of human MCL. Selective inhibition of PRMT5 in these models and MCL cell lines leads to disruption of constitutive PI3K/AKT signaling, dephosphorylation and nuclear translocation of FOXO1, and enhanced recruitment of this tumor suppressor protein to chromatin. We identified 136 newly emerged FOXO1-bound genomic loci following 48 hours of PRMT5 inhibition in the CCMCL1 MCL line by performing chromatin immunoprecipitation-seq analysis. These genes were markedly upregulated in CCMCL1 cells treated with the PRMT5 inhibitor PRT382 as determined by RNA-seq analysis. Among those genes, we identified and confirmed FOXO1 recruitment to the promoter of BAX, a pro-apoptotic member of the BCL2 family of proteins. Treatment of MCL cell lines (Granta-519, CCMCL1, Z-138, and SEFA) with the selective PRMT5 inhibitor PRT382 (10, 100nM) led to upregulation of BAX protein levels and induction of programmed cell death as measured by annexin V/PI staining and flow cytometry. We hypothesized that induction of BAX would trigger a therapeutic vulnerability to the BCL2 inhibitor venetoclax, and that combination PRMT5/BCL2 inhibitor therapy would drive synergistic cell death in MCL. Single agent and combination treatment with venetoclax and PRT382 was performed in eight MCL lines including a new cell line generated from our ibrutinib-refractory PDX model (SEFA) and IC50 and synergy scores were calculated. The Z-138 line was most sensitive to venetoclax (IC50<10nM) while CCMCL-1, SP53, JeKo-1, and Granta-519 demonstrated relative resistance (IC50>1uM). All lines reached an IC50 <1uM when co-treated with PRT382, with IC50 values ranging from 20 - 500nM. Combination treatments showed high levels of synergy (scores > 20) in 4 lines and moderate synergy (scores 10-20) in 2 lines. The two lines with the highest levels of synergy, Z-138 and SEFA, express high levels of BCL-2 and are Ibrutinib resistant. Overall there was a strong positive correlation between BCL2 expression and synergy score (r=0.707), and no correlation between PRMT5 expression and synergy score (r=0.084). In vivo evaluation in two preclinical MCL models (Granta-519 NSG mouse flank and an ibrutinib-resistant MCL PDX) showed therapeutic synergy with combination venetoclax/PRT382 treatment. In both models, mice were treated with sub-therapeutic doses of venetoclax and/or PRT543 (Granta) or PRT382 (IR-MCL PDX) and tumor burden assessed weekly via flank mass measurement (Granta) or flow cytometry (IR-MCL-PDX). Combination treatment with well-tolerated doses of venetoclax and PRMT5 inhibitors in both MCL in vivo models showed synergistic anti-tumor activity without evidence of toxicity. This preclinical data provides mechanistic rationale while demonstrating therapeutic synergy and lack of toxicity in this preclinical study and justifies further consideration of this combination strategy targeting PRMT5 and BCL2 in MCL in the clinical setting. PRT543, a selective PRMT5 inhibitor, has been advanced into clinical studies for the treatment of patients with solid tumors and hematologic malignancies, including MCL (NCT03886831). Disclosures Zhang: Prelude Therapeutics: Employment. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

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Combination Bortezomib (PS341, Velcade) and Rituximab Treatment Affects Multiple Survival and Death Pathways To Promote Apoptosis in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v106.11.2407.2407 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2407-2407 ◽

Cited By ~ 4

Author(s):

Navin Wadehra ◽

Teresa Lin ◽

Timothy Ryan ◽

Ashley Schneider ◽

Allison Pepple ◽

...

Keyword(s):

Animal Model ◽

Cell Death ◽

Combination Therapy ◽

Mantle Cell Lymphoma ◽

Combination Treatment ◽

Cell Lymphoma ◽

Proteasome Inhibition ◽

Mantle Cell ◽

Induction Of Apoptosis ◽

Induced Apoptosis

Abstract Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkin’s lymphoma that is associated with an aggressive clinical course and a particularly poor prognosis. The mechanisms that contribute to resistance of MCL to chemotherapy are not clear, however, recent work examining the consequences of ubiquitin-proteasome pathway inhibition on cell cycle (p21, p27) and key survival/death networks (NFkB, p53, Bcl2) has provided rationale for exploring combination regimens that include tumor-specific reagents (rituximab) and the 26S proteasome inhibitor bortezomib. In this study, we examined the effects of combination treatment with bortezomib and rituximab on MCL patient samples and three patient-derived cell lines (Jeko, Mino, SP53). Cells treated with bortezomib (10 – 100nM) for 4 hours demonstrated proteasome inhibition that persisted for 24 hours but returned to baseline activity at 48 hours after treatment. Despite transient proteasome inhibition, combination therapy with bortezomib (10–100nM for 4hrs) and rituximab (1 mg/ml immobilized with 20 mg/ml goat anti-human IgG) resulted in synergistic induction of apoptosis that persisted for as long as 72 hours after treatment. While bortezomib (100 nM) induced apoptosis in 18.3 ± 6.5% and rituximab induced apoptosis in 24.5 ± 4.5% of MCL cells, combination treatment resulted in 57.4 ± 5.1% apoptosis at 48 hours (p ≤ 0.02). Pretreatment of MCL cells with the broad spectrum caspase inhibitor zVAD-FMK (10 mM) showed that bortezomib-induced cell death occurred by caspase-dependent mechanisms, however, when immobilized rituximab was added, cell death occurred via caspase dependent and independent pathways. Single agent bortezomib (10 nM) or rituximab treatment of Mino and Jeko lines resulted in decreased levels of nuclear NFkB complex(s) capable of binding p65 consensus oligonucleotides (28% and 21% reduction, respectively), while combination treatment resulted in enhanced reduction of detectable nuclear NFkB (36% reduction, p ≤ 0.0007). Similar trends were observed with primary MCL cells. Experiments with an IKK inhibitor (PS1145, Millenium Pharmaceuticals) resulted in nuclear NFkB reduction without equivalent induction of apoptosis which led us to hypothesize that other pro-death pathways might be operable with combination treatment. Western blot analysis of BCL2-family members revealed that combination treatment of MCL lines resulted in near complete elimination of Bcl-xL protein while Bcl-2 protein levels remained unchanged. The pro-death gene product Bax was induced in a synergistic fashion with combined bortezomib and rituximab treatment. Finally, we have developed a reliable preclinical animal model utilizing the severe combined immune deficient (SCID) mouse engrafted with three patient-derived MCL cell lines. Each cell line results in a characteristic pattern of tumor burden and highly reproducible time to develop advanced disease. We are currently evaluating combination therapy with bortezomib and rituximab in this preclinical animal model. Our preclinical evaluation provides clear rationale for pursuing combination strategies that inhibit the proteasome in combination with tumor-specific immunotherapy in patients with MCL.

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Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v112.11.886.886 ◽

2008 ◽

Vol 112 (11) ◽

pp. 886-886 ◽

Cited By ~ 1

Author(s):

Lapo Alinari ◽

Erin Hertlein ◽

David M. Goldenberg ◽

Rosa Lapalombella ◽

Fengting Yan ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Preclinical Model ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

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FTY720 Increases CD74 Expression and Sensitizes Mantle Cell Lymphoma Cells to Milatuzumab-Mediated Cell Death

Blood ◽

10.1182/blood.v118.21.600.600 ◽

2011 ◽

Vol 118 (21) ◽

pp. 600-600

Author(s):

Lapo Alinari ◽

Emilia Mahoney ◽

John T. Patton ◽

Xiaoli Zhang ◽

Lenguyen Huynh ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Median Survival ◽

Combination Treatment ◽

Cell Lymphoma ◽

Mantle Cell

Abstract Abstract 600 Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death via down-modulation of phospho-Akt and Cyclin D1, and subsequent cell cycle arrest (1). However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. Here we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes, increased LC3-II and p62 levels. FTY720 is phosphorylated in vivo by sphingosine kinase 2 and converted to p-FTY720, which binds to sphingosine-1-phosphate (S1P) receptors. A non-phosphorylatable FTY720 derivative (OSU-2S) was recently developed at the Ohio State University (2): OSU-2S treatment induces MCL cell death and shows features of autophagy blockage that led us to conclude that FTY720 phosphorylation and its interaction with SP1 receptors are not required for FTY720-mediated cell death and blockage of autophagy in MCL cells. We also demonstrate that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. We have recently reported CD74 to be expressed on MCL cells and that milatuzumab (Immunomedics, Morris Plains, NJ), a humanized anti-CD74 monoclonal antibody, has significant anti-MCL activity in vitro and in vivo (3). This finding provided the rationale for examining combination therapy with FTY720 and milatuzumab. The in vitro survival of 4 MCL cell lines treated with FTY720, immobilized milatuzumab, and the combination was determined at 24 hours by Annexin-V/PI staining and flow cytometry. Incubation of 4 MCL cell lines with FTY720 and milatuzumab (1 μg/ml) resulted in a statistically significant decrease in cell viability compared to either agent alone for each of the four cell lines (P< 0.01), despite using FTY720 at concentrations lower than the LC50 previously published [Jeko-1 FTY720: 10 μM (LC50: 12.5 μM), Z-138 and UPN-1: 6 μM (LC50: 7.5 μM); Mino 3.75 μM (LC50: 7.5μM)] (1). Notably, combination treatment resulted in synergistic killing in cell lines derived from patients with blastoid variant MCL (Jeko-1, Z-138, UPN-1), despite the fact that both FTY720 and milatuzumab as single agents showed only modest activity. Incubation of primary tumor cells from 6 MCL patients (3 blastoid variant and 3 classic MCL) with FTY720 (2.5 μM, LC50: 5 μM) and miltauzumab induced an average 78.5% cell death compared to 47% of FTY720 treated cells and 50% the milatuzumab-treated cells (P=0.0005 and P=0.0014, respectively). To examine the in vivo activity of FTY720 and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice (n=10/group) were treated starting at day 15 post engraftment. Twenty control mice received either placebo (saline) or trastuzumab (15 mg/kg) treatment. The third group was treated with FTY720 (5 mg/kg) every day for 2 weeks via i.p injection. The fourth group received milatuzumab (15 mg/kg) every three days, via i.p. injection. The fifth group received the combination of FTY720 and milatuzumab. The median survival for the combination-treated group was 36 days (95% CI:31,36), compared to 28 days for the saline-treated mice (95% CI:24,31), 27 days for the trastuzumab-treated mice (95% CI:23,29), 31 days for the FTY720-treated mice (95% CI:28,32), and 33.5 days for the milatuzumab-treated mice (95% CI:23,34). The combination treatment significantly prolonged survival of this group compared to control groups (P<0.0001), FTY720 (P=0.0001) and milatuzumab (P=0.0048). The most clinically relevant aspect of these findings is that we demonstrate that a potent anti-MCL agent (FTY720) has also the ability to modulate a druggable target (CD74) by preventing its degradation in the autophagic-lysosomal pathway. We believe these findings support clinical evaluation of this combination in patients with MCL. Disclosures: Off Label Use: fty720 immunosupressive drug milatuzumab fully humanized anti-CD74 monoclonal antibody. Goldenberg:Immunomedics: Equity Ownership, Immunomedics owns milatuzumab, Patents & Royalties.

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