Chimerized Anti-ICOS 314.8 Monoclonal Antibodies Inhibit Tumor Cells and Regulatory T Cells in Patients with Sézary Syndrome

Abstract Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy. Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®). Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry. Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS. Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered. ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1). Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031). Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p<0.01; CI95%:-53--27). The efficacy was better than with alemtuzumab, but there was no significant difference with moga. The ADCP effect induced by anti-ICOS did not differ with moga. Interestingly, anti-ICOS were effective in 6 moga-resistant patient, as they induced 38.9±5.9% of apoptosis, compared to 12.4±4.7% with moga (p<0.001). Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab. Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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ICOS Is Widely Expressed in Cutaneous T-Cell Lymphoma and Its Targeting Promotes Potent Killing of Malignant Cells

Blood ◽

10.1182/blood-2021-150677 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 790-790

Author(s):

Florent Amatore ◽

Nicolas Ortonne ◽

Marc Lopez ◽

Mathilde Barré ◽

Florence Orlanducci ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Lines ◽

Early Stage ◽

Cd4 Cells ◽

Advisory Committees ◽

T Cell Lymphomas ◽

Sézary Cells ◽

Privately Held

Abstract Background: Advanced cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), do not deliver significant long-term improvements in patient outcomes. More recently, mogamulizumab and anti-KIR3DL2 provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest. Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. Skin samples from 12 patients with B-cell lymphomas, 14 with CD30 + lymphoproliferative disease (LPD), 12 with primary cutaneous CD4+ small/medium T-cell lympho-proliferative disorder and 13 with angioimmunoblastic T-cell lymphoma (AITL) were used as controls. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes. Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS 314.8 monoclonal antibodies with MMAE and pyrrolobenzodiazepine (PBD), in comparison to BV. We used ICOS + CTCL cell lines (MyLa, MJ and HUT78), murine xenograft models with MyLa and ICOS + Patient Derived Xenografts (PDXs) from patients with SS and AITL. In order to identify the best anti-ICOS clone that we should develop for a clinical trial, we evaluated the affinity of the antibody on the receptor, the internalization capacity of the antibody using pHAb Reactive Dyes kit (Promega®), and the ability of the antibody to act as an ADC using a secondary conjugate (Mab-ZAP kit, Advanced Targeting Systems®). Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in all the involved nodes. Double staining experiments which were performed in both skin and lymph node revealed that ICOS expression appears mainly restricted to neoplastic CD4 + T-cells, with rare ICOS +CD8 + T-cells in the tumor micro-environment. ICOS expression by circulating Sézary cells was strong: 69 ± 7.3% versus 38.8 ± 7.1% of non-tumoral CD4 + cells (p<0.009; CI95%: 8.7-51.6); and 31 ± 3.2% of CD4+ cells in HD (p<0.0001; CI95%: 20.3-46.3). Percentages of ICOS + Tregs were significantly higher in patients with SS than in HD. In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS-MMAE (IC50 = 8.2ng/mL) and anti-ICOS-PBD (IC50 = 1.2ng/mL) ADCs. In a mouse xenograft model (MyLa), anti-ICOS-MMAE ADCs provided a longer overall survival (OS) than BV (HR=15.2; CI95%: 3.2-71.1; p<0.0006). Finally, in ICOS + PDXs, anti-ICOS-MMAE ADCs significantly improved OS, and reduced the number of tumor cells in the blood, spleen, and bone marrow. No evidence of ADC toxicity was observed in treated mice. Among 8 different anti-ICOS clones, clone 314.8 had the best affinity on MyLa and MJ cell lines. Clones 53.3, 293.1, 92.17, 88.2 and 279.1 also had good affinity to receptor, whereas clones 145.1 and 121.4 had poor affinity. Using the internalization pHAb reactive dyes kit, we found that clones 314.8, 53.3, 92.17, 88.2 internalized significantly better and faster than the other ones. In order to verify if there is a correlation between internalization capacity and ADC effect, clones 53.3, 92.17 and 145.1 were coupled to MMAE. While anti-ICOS-53.3 and anti-ICOS-92.17 ADCs had similar efficacy to anti-ICOS-314.8 ADCs on MyLa, anti-ICOS 145.1 ADCs resulted in significantly lower cell death. Finally, all clones showed good ability to act as ADCs with Mab-ZAP, excluding clones 279.1, 145.1 and 121.4. Discussion: ICOS is a promising therapeutic target because it is expressed both by tumor T-cells and regulatory T-cells. We report for the first time the strong and frequent expression of ICOS in CTCLs, as well as the preclinical efficacy of anti-ICOS ADCs on CTCL cell line and PDXs. These results could be extended to the spectrum of follicular variant peripheral T-cell lymphomas. Conclusion: Collectively, our findings provide the preliminary basis for a therapeutic trial Figure 1 Figure 1. Disclosures Lopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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Targeting BTN2A1 By a Unique Activating Mab Improves Vγ9Vδ2 T Cell Cytotoxicity Against Primary Acute Lymphoblastic Blasts

Blood ◽

10.1182/blood-2021-152577 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2302-2302

Author(s):

Anne-Charlotte Le Floch ◽

Caroline Imbert ◽

Aude De Gassart ◽

Florence Orlanducci ◽

Aude Le Roy ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Leukemic Cells ◽

Target Cells ◽

Advisory Committees ◽

Vγ9vδ2 T Cells ◽

Privately Held

Abstract Introduction Vγ9Vδ2 T cells are new promising cytotoxic effectors in hematological malignancies. In acute myeloid leukemia and in non-Hodgkin lymphomas, Vγ9Vδ2 T cells-based immunotherapy has shown encouraging results both in preclinical models and in early phase clinical trials. Acute lymphoblastic leukemia (ALL) includes very heterogeneous clinico-biological entities, for which recent immunotherapy approaches are currently being developed. Nevertheless, global prognosis of ALL patients still be poor with a 5 years-overall survival of less than 40% and therefore, treatments need to be improved. Very few data are currently available on susceptibility of ALL blasts to Vγ9Vδ2 T cell cytotoxic activity. Vγ9Vδ2 T cells are activated by phosphoantigens bound to BTN3A1 on target cells. BTN3A molecules are targeted at clinical level, with the ICT01 agonist monoclonal antibody (mAb), that is currently tested in a multicentric phase ½ study (EVICTION study). Biology of Vγ9Vδ2 T cells has recently undergone a new paradigm with the identification of BTN2A1 as the direct ligand for Vγ9 chain of γδ TCR. BTN2A1 is mandatory for Vγ9Vδ2 T cell activation but its precise role in modulating functions of Vγ9Vδ2 T cells remains unknown. Here, we show that allogenic and autologous Vγ9Vδ2 T cells exert cytolytic functions against ALL cell lines and primary ALL blasts, and we report that Vγ9Vδ2 T cell cytotoxic activity is enhanced after treatment with a unique agonist mAb targeting BTN2A1. Material and methods 5 ALL cell lines (697, RS4;11, NALM-6, HPB-ALL, SUP-T1) and PBMC from 11 adults ALL patients at diagnosis (B-ALL, T-ALL and Ph+ ALL) were tested in functional assays. We evaluated apoptosis of ALL cell lines and of primary ALL blasts after coculture with allogenic Vγ9Vδ2 T cells. ALL samples were also tested for their expansion capacities and a degranulation assay was performed at D14. We assessed in parallel relative quantification of the level expression of BTN2A1 (ICT0302 and 7.48 epitopes), and BTN3A (20.1 and 108.5 epitopes) on surface of ALL blasts. DAUDI-BTN2AKO+2A1 and HEK293-BTN2AKO+2A1 cells were used in binding assays, and modulation of TCR binding was assessed using recombinant tetramerized Vγ9Vδ2 TCR. Results We showed that Vγ9Vδ2 T cells exert spontaneous cytotoxicity against ALL cell lines and primary ALL blasts with a heterogeneous susceptibility depending on the target. We demonstrated that anti-BTN2A1 ICT0302 agonist mAb significantly enhanced Vγ9Vδ2 T cells mediated apoptosis in comparison to control condition, even for the less spontaneously susceptible cells. We confirmed these observations with degranulation of autologous Vγ9Vδ2 T cells expanded from 5 ALL patients at diagnosis that was increased after treatment with anti-BTN2A1 ICT0302 agonist mAb. BTN3A and BTN2A1 were detected on surface of ALL blasts, and BTN3A 108.5 was the most expressed epitope. Interestingly, we observed that anti-BTN2A1 ICT0302 strongly increased binding of a recombinant Vγ9Vδ2 TCR to target cells using with HEK293 and DAUDI cells. Discussion Our results highlighted that Vγ9Vδ2 T cells exert cytolytic functions against ALL cells, both in allogenic and autologous setting and demonstrated that BTN2A1 targeting with our unique agonist mAb could potentiate effector activities of Vγ9Vδ2 T cells against ALL blasts. These results indicate that the sensitization of leukemic cells can be induced by activation BTN3A as well as BTN2A1 mAbs. These data bring novel understanding on the biology of BTN2A1 on leukemic cells and our ability to enhance both binding and function. These findings could be of great interest for the design of innovative Vγ9Vδ2 T cells-based immunotherapy strategies for treating ALL that could be extended to other cancer types. Disclosures De Gassart: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Cano: ImCheck Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company. Olive: Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Anti-BTN2A1 ICT0302 is a murine agonist monoclonal antibody targeting BTN2A1 whose aim is to increase Vgamma9Vdelta2 T cells functions.

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A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.1841.1841 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1841-1841

Author(s):

Dharminder Chauhan ◽

Ajita V. Singh ◽

Arghya Ray ◽

Teru Hideshima ◽

Paul G. Richardson ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Inhibitory Effect ◽

Advisory Committees ◽

Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

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Dual Inhibition of Canonical and Non-Canonical NF-KB Pathway Demonstrates Significant Anti-Tumor Activities in Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v118.21.2902.2902 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2902-2902

Author(s):

Claire Fabre ◽

Naoya Mimura ◽

Kathryn Bobb ◽

Gullu Gorgun ◽

Diana D. Cirstea ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Inhibitory Effect ◽

Growth Inhibitory Effect ◽

Advisory Committees ◽

Growth Inhibitory ◽

Canonical Pathways ◽

Dual Inhibition

Abstract Abstract 2902 NF-kB plays a crucial role in the pathogenesis of multiple myeloma (MM). In MM cells, NF-kB pathway is constitutively activated and regulates transcription of genes whose protein products mediate proliferation, survival and drug resistance. In the context of the bone marrow (BM) microenvironment, NF-kB modulates the expression of cytokines (ie, IL6, TNFalpha) and adhesion molecules (ie, ICAM-1). Importantly, these cytokines and adhesion to BM stromal cells (BMSCs) further activate NF-kB pathway. Previous studies have shown that both canonical and non-canonical pathways contribute to total NF-kB activity in MM cells. Therefore inhibition of both pathways is necessary to target NF-kB. However, current therapeutic strategies can only inhibit the canonical, but not the non-canonical pathway. In this study, we examined the biologic impact of dual inhibition of both canonical and non-canonical pathways in MM cells using a novel small molecule inhibitor PBS-1086 (Profectus BioSciences) which selectively inhibits binding of Rel family member proteins to DNA. Importantly, the binding activity of all Rel family member proteins (RelA, RelB, NF-kB1, NF-kB2, cRel) to DNA was inhibited by PBS-1086, confirming that PBS-1086 blocks both canonical and non-canonical pathways in MM cell lines. We first investigated growth inhibitory effect of PBS-1086 in vitro. PBS-1086 potently inhibited the growth of MM cell lines (MM1S, MM1R, INA6, LR5, Dox40, KMS18, RPMI-8226 and U266) in a dose-dependent fashion with IC50 ranges of 0.15–5 μM. In contrast, PBS-1086 showed modest cytotoxicity on normal peripheral blood mononuclear cells from healthy volunteers. Similar growth inhibitory effect were observed in CD138+ primary tumor cells derived from MM patients. PBS-1086 induced apoptosis in MM1S cell line in a time-dependent manner, evidenced by annexin V-PI staining by flow cytometry and cleaved caspase 8, 9, 3 and PARP, suggesting that PBS-1086 activates both extrinsic and intrinsic apoptotic pathways. Importantly, PBS-1086 can overcome the proliferative and anti-apoptotic effects of BMSCs, associated with inhibition of NF-kB activity. We next examined the combination effect of PBS-1086 with other agents. PBS-1086 with bortezomib synergistically enhanced anti-MM activity even in bortezomib-resistant cell lines (Dox40, ANBL6-VR5) and primary tumor cells from MM patients. Finally, we investigated the effect of PBS-1086 in vivo in a murine xenograft model of human MM cells. Tumor-bearing mice were divided into 6 groups: non-treated, vehicle control, PBS-1086 (7.5 mg/kg ip daily), bortezomib (0.5 mg/kg IV, twice weekly) and the combination of PBS-1086 (either at 2.5 mg/kg or 7.5 mg/kg) with bortezomib. PBS-1086 showed significant anti-MM activity in combination (2.5 and 7.5 mg/kg) groups versus control group (p =0.00039 and p =0.00084, respectively). Combination groups also had significantly (p < 0.05) prolonged survival compared to single agent treatment group (PBS-1086 or bortezomib). In conclusion, our preclinical studies show that PBS-1086 is a promising novel therapeutic agent and our data supports further clinical evaluation of this agent in combination with bortezomib for the treatment of MM. Disclosures: Bobb: Profectus BioSciences: Employment; Rel-MD: Employment. Zhang:Profectus BioSciences: Employment; Rel-MD: Employment. Meshulam:Profectus BioSciences: Employment; Rel-MD: Employment. Mitsiades:Millennium: Consultancy, Honoraria. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Hideshima:Acetylon: Consultancy. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees.

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Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001

Blood ◽

10.1182/blood.v124.21.4712.4712 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4712-4712 ◽

Cited By ~ 5

Author(s):

Deepika Sharma Das ◽

Ze Tian ◽

Arghya Ray ◽

Durgadevi Ravillah ◽

Yan Song ◽

...

Keyword(s):

Oxidative Stress ◽

Clinical Trial ◽

Drug Resistance ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Advisory Committees ◽

Healthy Donors

Abstract Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA. Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance. Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib

Blood ◽

10.1182/blood.v118.21.2745.2745 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2745-2745 ◽

Cited By ~ 1

Author(s):

Deborah L. White ◽

Liu Lu ◽

Timothy P. Clackson ◽

Verity A Saunders ◽

Timothy P Hughes

Keyword(s):

Tyrosine Kinase ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Kinase Inhibitor ◽

K562 Cells ◽

Temperature Dependent ◽

Advisory Committees ◽

Significant Difference

Abstract Abstract 2745 Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210). While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NA To further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM's (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1. These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures: White: BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Follicular Lymphoma Grade 3A Exhibits Pathological and Cytogenetic Diversity, but Similar Prognosis Compared to FL Grade 1-2: Pooled Analysis of 1757 Follicular Lymphomas from the PRIMA and Relevance Trials

Blood ◽

10.1182/blood-2018-99-115831 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 778-778

Author(s):

Camille Laurent ◽

Danielle Canioni ◽

Bettina Fabiani ◽

Véronique Meignin ◽

Catherine Chassagne-Clément ◽

...

Keyword(s):

Follicular Lymphoma ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Statistical Difference ◽

Prognostic Significance ◽

Advisory Board ◽

Genomic Diversity ◽

Advisory Committees ◽

Significant Difference

Abstract Introduction: Follicular lymphoma (FL) is graded as 1, 2, 3A and 3B based on the number of centroblast cells. Although FL grade 3A is considered as a low-grade lymphoma, its prognostic significance compared to FL grade 1-2 remains controversial, especially for particular morphological variants with large cleaved cells or blastoid features, which are not recognized as a specific entity. Method: In order to clarify these points, FL grade 3 patients (pts) were selected from a large series of 2247 untreated pts enrolled in two LYSA trials: PRIMA (evaluating rituximab (R) maintenance after R-chemotherapy) and RELEVANCE (evaluating lenalidomide plus rituximab (R2) followed by R2 maintenance versus R-chemotherapy). FL3B pts were excluded from both trials. Sufficient material and clinical informations were available for 1757 out of 2247 pts. Among them, 161 pts including 88 of 950 PRIMA pts (9.2%) and 73 of 734 RELEVANCE pts (9.2%) were classified as FL3A. Among these FL3A cases, a panel of 7 expert hematopathologists identified 48 cases (2.7% of 1757 analyzed FL cases) which contained a significant component of large cleaved cells or medium-sized blastoid cells but did not meet grade 3B criteria. These cases were called FL3 "unclassified" (FL3U) as compared to classical FL3A (cFL3A) cases. We then analyzed the correlations between the histologic grade, phenotypic/cytogenetic features and clinical outcome. Results: FL3U were characterized by: i) proliferation of large cleaved tumor cells with moderately coarse to fine chromatin and absent or inconspicuous nucleoli (n=30) or predominance of medium-sized blastoid tumor cells with fine chromatin and small nucleoli (n=18) and ii) expression of CD20 and at least one germinal center marker. Mean of MYC and MUM1 protein expression in FL3U were 18% [from 3 to 25%] and 14.8% [3-35%], respectively, with no significant difference with cFL3A. Ki67 expression was higher in FL3U than in cFL3A (55.6% [20-90%] vs 41.3% [2-85%]) (p=0.008). Expression of p53 protein was slightly higher in FL3U than in cFL3A (37% [10-80%] vs 34.9% [8-90%] (p=0.052). FL3U had a tendency to harbor less frequent BCL2 rearrangements than cFL3A (74% vs 95%) (p=0.0620), whereas BCL6 rearrangements were significantly higher in FL3U than in cFL3A (29% vs 0%) (p=0.0034). The frequency of MYC rearrangements and 1p36 deletions in FL3U (9.6% and 6% of FL3Us, respectively) showed no significant difference with cFL3A. No detectable alteration in IRF4 locus was seen in both FL3U and cFL3A. The median age of FL3U pts was 58 years with 1:1 ratio of males to females and most pts had advanced stage at diagnosis with frequent marrow infiltration and intermediate to high FLIPI score. Outcome of pts with FL3U was not significantly different to that of cFL3A pts in Cox multivariate analyses. After a median follow-up of 117 months for PRIMA and 38 months for RELEVANCE, 89.6% of FL3U and 85.2% of cFL3A were alive with no significant difference between the 2 groups (p=0.4507). There was also no statistical difference in progression free survival (PFS) between the 2 groups (p= 0.1479). Similarly, we did not found any statistical difference in PFS between FL3U and FL1-2 and between FL1-2 and cFL3A (p=0.9210 and p=0.5375, respectively); as well as in overall survival (OS) (p=0.6223 and p=0.0960, respectively). Finally, the outcome of the whole group of FL3A pts including cFL3A and FL3U variants was similar to FL1-2 pts. Conclusion: FL grade 3A exhibits pathological and genomic diversity due to FL3U variants characterized by higher amounts of medium-sized blastoid or large cleaved cells, higher Ki67 proliferative index and p53 expression, together with increased frequency of cytogenetic BCL6 alterations and lower frequency of BCL2 rearrangements. However, in both PRIMA and RELEVANCE trials, FL3U pts showed no significant difference in terms of PFS or OS as compared to both FL1-2 and cFL3A pts. These results indicate that FL3A represents a spectrum of proliferations with variable maturity, proliferative activity and genomic alterations that may be intermediate points of progression toward FL3B/transformation. They suggest that the distinction between cFL3A and FL3U variant, as well as between FL1-2 and FL3A may not have any prognostic significance using modern rituximab- or lenalidomide-based treatments, although this has to be confirmed with different drug combinations. Disclosures Cartron: Gilead Sciences: Honoraria; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Sanofi: Honoraria; Janssen: Honoraria. Morschhauser:Epizyme: Consultancy; Janssen: Other: Scientific Lectures; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Salles:Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Pfizer: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Novartis: Consultancy, Honoraria; Servier: Honoraria, Other: Advisory Board; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BMS: Honoraria, Other: Advisory Board; Morphosys: Honoraria; Gilead: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Merck: Honoraria; Takeda: Honoraria; Servier: Honoraria. Xerri:Janssen: Other: Travel.

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Validation of NMT1 and NMT2 As Novel Drug Targets in Adult Acute Myeloid Leukemia: Rationale for N-Myristoyltransferase Inhibition with Pclx-001 for Clinical Trials

Blood ◽

10.1182/blood-2021-154000 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4344-4344

Author(s):

Jay Gamma ◽

Aishwarya Iyer ◽

Megan Yap ◽

Zoulika Zak ◽

Krista Vincent ◽

...

Keyword(s):

Clinical Trials ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Cell Lines ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Research Funding ◽

Advisory Committees ◽

Acute Myeloid ◽

Privately Held

Abstract Background: N-terminal myristoylation is the transfer of the saturated fourteen carbon fatty acid myristate to an N-terminal glycine residue. This co- or post-translational protein modification promotes protein-protein and protein-lipid interactions and is essential for proper membrane localization and/or activity of up to 600 human intracellular proteins. N-myristoyltransferases (NMTs) are the enzymes responsible and two isoforms are found in humans. NMT1 (ubiquitous and essential for cell survival) and NMT2 (more variably expressed) differ in activity level and substrate specificities. NMT expression levels vary in some cancers, and with myristoylation being essential for activity of certain oncogenes including Src Family Kinases (SFKs). NMTs have therefore been proposed as an anti-cancer target. Dysregulation and oncogenic activity of SFKs occurs frequently in acute myeloid leukemia (AML), suggesting NMT inhibition could provide therapeutic benefit. PCLX-001 is a low nanomolar small molecule pan-NMT inhibitor with high oral bioavailability in clinical trials as once daily oral therapy for lymphoma and solid tumors. Methods and Results: Data from the TCGA Transcriptome database showed high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML, and high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, as well as reduced levels of SFK proteins and SFK phosphorylation. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations which spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. AML cell lines showed significant increase in BIP protein and ER stress in response to PCLX-001, along with caspase 3 cleavage. In an AML cell line derived xenograft (CDX) and two AML patient derived xenograft (PDX) series (n=1 DX for MV-4-11 and n=2 PDX), PCLX-001 monotherapy had dose-dependent anticancer activity and resulted in complete remissions in subcutaneous AML cell deposits. In tail-vein injection PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. Conclusions: These findings validate NMT inhibition as a novel therapeutic strategy for AML. PCLX-001 preferentially targeted AML cells that rely on oncogenic activity of myristoylated proteins, inducing apoptosis and reducing leukemic burden. PCLX-001 warrants evaluation in clinical trials for adult AML. Disclosures Gamma: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company. Yap: Pacylex Pharmaceuticals: Current holder of individual stocks in a privately-held company, Patents & Royalties. Beauchamp: Pacylex Pharmaceuticals: Current Employment, Current holder of individual stocks in a privately-held company, Patents & Royalties. Mackey: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company. Pemmaraju: Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Aptitude Health: Consultancy; Sager Strong Foundation: Other; Celgene Corporation: Consultancy; LFB Biotechnologies: Consultancy; Plexxicon: Other, Research Funding; MustangBio: Consultancy, Other; Roche Diagnostics: Consultancy; Daiichi Sankyo, Inc.: Other, Research Funding; DAVA Oncology: Consultancy; Cellectis S.A. ADR: Other, Research Funding; Springer Science + Business Media: Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Samus: Other, Research Funding; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; CareDx, Inc.: Consultancy; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Incyte: Consultancy; Affymetrix: Consultancy, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Borthakur: Astex: Research Funding; Ryvu: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; ArgenX: Membership on an entity's Board of Directors or advisory committees; University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Brandwein: AbbVie: Honoraria; Jazz: Honoraria; Taiho: Honoraria; Astellas: Honoraria; Bristol Myers Squibb: Honoraria; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Berthiaume: Pacylex Pharmaceuticals, Inc.: Current holder of individual stocks in a privately-held company.

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Cells of the Osteoblast Lineage Confer Myeloma Cell Resistance to Established and Investigational Therapeutic Agents

Blood ◽

10.1182/blood.v120.21.3995.3995 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3995-3995

Author(s):

Catriona A. Hayes ◽

Paul Dowling ◽

Richard W.J. Groen ◽

Douglas W. McMillin ◽

Jake E. Delmore ◽

...

Keyword(s):

Tumor Cell ◽

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Research Funding ◽

Kinase Inhibitors ◽

Cell Contact ◽

Advisory Committees ◽

Direct Cell ◽

Osteoblast Lineage

Abstract Abstract 3995 Introduction: Multiple myeloma (MM) cells suppress osteoblast (OB) maturation and function and induce osteoclast-mediated bone resorption. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present in the MM bone microenvironment and their impact on MM cell response to treatment has not been fully characterized. We therefore studied human immortalized cells of the osteoblast lineage as surrogate models for in vitro studies on the effects of pre-osteoblasts on MM cell proliferation, survival and drug resistance. Methods: Immortalized hFOB 1.19 (here referred to as hFOB) and HOBIT cells were utilized in our studies, because, in the absence of Dexamethasone or vitamin D, they express markers consistent with pre-osteoblast stage of differentiation (high type I collagen levels; and low alkaline phosphatase, osteocalcin and osteopontin expression). Monolayer cultures of hFOB cells were performed to determine drug concentrations that did not exhibit cytotoxic effects on these pre-osteoblasts. Next, co-cultures of hFOB and HOBIT cells were performed using a panel of tumor cell lines from MM (n=13), breast (n=1) and lung (n=1) cancer. Compartment-specific bioluminescence imaging (CS-BLI) assays were undertaken to quantify the proliferative responses of the tumor cells in the presence of pre-osteoblasts, and if any changes are observed in tumor cell responses to established or novel therapeutic agents. Assays with Transwell insert chambers were conducted to allow for culture of tumor cells proximate to pre-osteoblasts, but preclude their direct cell-to-cell contact, in order to determine whether paracrine factors released by pre-osteoblasts are sufficient to recapitulate the effect(s) of their co-culture with tumor cells. Finally, cytokine or cytokine receptor neutralization studies with monoclonal antibodies or selective kinase inhibitors were performed to investigate the functional contribution of IL-6 or IGF1R-mediated signalling in tumor-pre-osteoblast interactions. Results: Pharmacologically relevant doses of conventional and novel therapies did not exhibit substantial cytotoxic effects on hFOB cells. Within 48hrs of co-culture, pre-osteoblasts stimulated proliferation of several tumor cell lines, including MM.1S, MM.1R, RPMI8226 and Dox40 in co-culture with hFOB; and NCI-H929, OPM2, H23 and Dox40 with HOBIT co-culture. hFOB cells decreased the sensitivity of 8 MM cell lines to at least one established (doxorubicin, dexamethasone, lenalidomide) or novel (vorinostat, pomalidomide) therapy tested. These observations were particularly pronounced in MM.1S and KMS34 cell lines. Subsequent Transwell-based co-culture assays showed that lack of direct cell-to-cell contact significantly decreases or even completely abrogates the resistance observed in respect to doxorubicin, lenalidomide or vorinostat in mono-cultures of tumor cells with pre-osteoblasts. Finally, IL-6 neutralization with monoclonal antibody did not overcome the proliferative effect of hFOB on MM cells. The anti-MM effect of IGF1R kinase inhibitors was also suppressed by co-culture with hFOB cells. These 2 latter results taken together indicate that the protection provided by hFOB cells is independent of IL-6 and IGF-1R-signaling in tumor or pre-osteoblasts. Conclusion: Our results suggest that cells of the osteoblast lineage may play a role as promoters of MM cell survival and resistance to diverse established and investigational therapeutics. Ongoing mechanistic and functional validation studies aim to delineate new therapeutic strategies to overcome pre-osteoblast-mediated drug resistance in MM and, potentially, other neoplasias. Disclosures: Richardson: Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees; Oncopep: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor Other.

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Synergistic Anti-Myeloma Activity of a Proteasome Inhibitor Marizomib and IMiD® Immunomodulatory Drug Pomalidomide

Blood ◽

10.1182/blood.v124.21.2099.2099 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2099-2099

Author(s):

Deepika Sharma Das ◽

Durgadevi Ravillah ◽

Arghya Ray ◽

Yan Song ◽

Paul G. Richardson ◽

...

Keyword(s):

Board Of Directors ◽

Tumor Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Response To Treatment ◽

Low Doses ◽

Advisory Committees ◽

Healthy Donors

Abstract Background and Rationale: Proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that a novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities (Chauhan et al., Cancer Cell 2005, 8:407-419). We also showed that marizomib triggers synergistic anti-MM activity in combination with lenalidomide (Chauhan et al., Blood 2010, 115:834-45). Pomalidomide, like lenalidomide, is an analogue of thalidomide with potent immunomodulatory activity, and has been approved by FDA for treatment of RRMM patients who have received at least two prior therapies including lenalidomide and bortezomib and showed disease progression on or within 60 days of completion of the last therapy. Approval of treatment is based on progression-free survival. Here we utilized in vitro and in vivo models of MM to examine the anti-MM activity of combined marizomib and pomalidomide. Materials and Methods:MM celllines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of marizomib and pomalidomide. Cell viability, apoptosis, and migration assays were performed using WST/MTT, Annexin V staining, and Transwell Inserts, respectively. Synergistic/additive anti-MM activity was analyzed by isobologram analysisusing “CalcuSyn” software program. Proteasome activity was measured, as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. MM.1S-tumor-bearing mice were treated with vehicle control, marizomib, pomalidomide or marizomib plus pomalidomide at the indicated doses for 21 days on a twice-weekly schedule for marizomib and 4 consecutive days weekly for pomalidomide. Statistical significance was determined using a Student’s t test. Pomalidomide was purchased from Selleck chemicals, USA; and marizomib was obtained from Triphase Inc., USA. Results: MM cell lines (MM.1S, MM.1R, INA-6, RPMI-8226, Dox-40, U266, LR5, ANBL6.WT, and ANBL6.BR) and primary patient MM cells were pretreated with DMSO control or with pomalidomide for 24h; marizomib was then added for an additional 24h, followed by assessment of cell viability. A significant decrease in viability of all cell lines and patient cells was observed in response to treatment with combined low doses of marizomib and pomalidomide, compared with either agent alone. Isobologram analysis confirmed the synergistic anti-MM activity of these agents (CI < 1.0). Tumor cells from 5 of 7 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, the cytotoxicity of combination therapy was observed in MM cell lines sensitive and resistant to conventional (dex, doxorubicin, melphalan) and novel (bortezomib) therapies. No significant decrease in viability of PBMCs from normal healthy donors was observed in response to treatment with combined low doses of marizomib and pomalidomide, suggesting selective anti-MM activity and a favorable therapeutic index for this combination regimen. Furthermore, marizomib plus pomalidomide inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies showed that marizomib plus pomalidomide-induced apoptosis was associated with: 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) downregulation of Cereblon, IRF4, c-Myc, and Mcl-1; and 3) enhanced inhibition of chymotrypsin-like, caspase-like and trypsin-like proteasome activities versus single agent alone. Furthermore, combined low doses of marizomib and pomalidomide blocked migration of MM cells and angiogenesis. In vivo studies using a subcutaneous human MM xenograft models show that combined low doses of marizomib and pomalidomide are well tolerated, inhibit tumor growth, and prolong survival. Conclusion: Our preclinical studies in MM disease models support a clinical trial of combined marizomib and pomalidomide to improve outcome in patients with relapsed and refractory MM. Disclosures Richardson: Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Trikha:Triphase Accelerator: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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