Selective HDAC6 Inhibition Via ACY-1215, Either Alone or in Combination with Bortezomib, Restores Osteoblast Function and Suppresses Osteoclast Differentiation in Multiple Myeloma

Abstract Abstract 2908 Bone disease in multiple myeloma (MM) is due to the disruption of the delicate balance between osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption. Agents that target both tumor cells and restore normal bone homeostasis can improve long-term disease control and prolong MM patient survival. It has been demonstrated that in vitro pan HDAC inhibitors accelerate OB maturation and suppress OC maturation, while bortezomib triggers OB activation and inhibits osteoclastogenesis. However it has recently been shown that vorinostat (SAHA), a non-selective HDAC inhibitor, causes bone loss in vivo by inhibiting immature OB. Here, we evaluated effects of a selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib, on MM cell growth and related bone disease. ACY-1215 in combination with bortezomib has synergistic cytotoxicity due to simultaneous inhibition of the proteasome and aggresome pathways. We confirm the in vivo anti-MM activity of ACY-1215 in combination with bortezomib in two different xenograft mouse models: human MM injected subcutaneously; and luciferase-expressing human MM injected intravenously (disseminated MM model). Tumor growth was significantly delayed and overall host survival significantly prolonged in animals treated with combined therapy (34 vs 22 days, n=7, p<0.0011) in plasmacytoma model and (40 vs 17 days, n=12, p<0.0001) in disseminated model. Importantly, we show that ACY-1215 alone and in combination with bortezomib overcomes the proliferative effect of bone marrow stromal cells (BMSCs) and cytokines. MM cells stimulate OC formation and function, while inhibiting OB differentiation via both cell-to-cell contact and cytokine secretion. Therefore, osteoclastogenesis is an important therapeutic target in MM. In this context, we evaluated the effect of ACY-1215 (1μM) and bortezomib (2.5nM) on OCs generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B ligand (RANKL). ACY-1215 alone and in combination with bortezomib inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, ACY-1215 (1μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines. We next examined the effect of ACY-1215, alone and in combination with bortezomib, on downstream targets in RANKL/RANK signaling. ACY-1215 plus bortezomib inhibits transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS and NFATC1. Since there is decreased OB function and new bone formation in MM, we next assessed the effect of ACY-1215 on OB differentiation. ACY-1215, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase enzyme activity and alizarin red staining. In addition, we show increased mRNA expression of b-catenin, osteocalcin, Runx2 and Sp7 (OB differentiation markers) in immature OB triggered by ACY-1215. Finally, ACY-1215 was not toxic to PHA stimulated PBMCs, suggesting a favorable side effect profile and therapeutic index. Our studies therefore demonstrate that ACY-1215, alone and in combination with bortezomib, can inhibit osteoclastogenesis enhance osteoblastogenesis, and inhibit MM cell growth. Based upon these studies, ongoing clinical trials are examining the efficacy of ACY-1215 in relapsed MM and associated bone disease. Disclosures: Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Bradner:Acetylon Pharmaceuticals, Inc.: Consultancy. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals, Inc.: founder; Merck: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

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Dysfunctional HDAC8 Impacts Genomic Integrity and Is a Novel Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153382 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1610-1610

Author(s):

Zuzana Chyra ◽

Srikanth Talluri ◽

Rao Prabhala ◽

Mehmet K. Samur ◽

Anil Aktas-Samur ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Hdac Inhibitors ◽

Dependent Manner ◽

Dna Breaks ◽

Advisory Committees

Abstract The histone modifications and associated changes in chromatin structure and function have emerged as important epigenetic mechanisms impacting gene expression and have significant translational relevance in cancers, including multiple myeloma (MM). Epigenetic intervention with histone deacetylases (HDACs) inhibitors is emerging as a promising therapeutic strategy in combination with current anti-myeloma agents. Although pan-HDAC inhibitors have been shown to be effective both in preclinical and clinical setting, they seem to be associated with toxicity. It is, therefore, extremely important to understand the biological and molecular roles of individual HDACs to then selectively target them to limit toxicities observed with pan-HDAC inhibitors. Based on our observation that elevated HDAC8 expression correlates with poor overall survival in MM patients in three different datasets including one publicly available dataset (GSE39754), we evaluated its functional role in MM. HDAC8, a member of class I HDAC isoenzymes, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones as well as non-histone proteins. We performed genetic modulation of HDAC8 by loss-of-function studies, using shRNA as well as siRNAs targeting HDAC8. Downregulation of HDAC8 in 3 different MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with induction of cell apoptosis. Consistently, treatment with a selective and potent HDAC8 inhibitor (OJI-1) caused a significant inhibition of MM cell growth in a panel of 20 MM cell lines (IC50 = 80 nM) in a time- and dose-dependent manner, while having a minimal impact on six PBMC samples from healthy donors both in resting and activated state (IC50 = 150 nM). The mechanism of cell death was apoptosis as demonstrated by annexin-labeling. Importantly, both the HDAC8 knockdown and OJI-1 treatment inhibited DNA breaks as evidenced from γH2AX expression or a single cell gel electrophoresis method to visualize and quantitate DNA breaks. HDAC8 inhibition also caused inhibition of RAD51 foci and HR activity, as measured by strand-exchange assay. Interestingly, non-homologous end joining in MM cells was not impacted by these treatments. Consistent with these data, the overexpression of HDAC8 in MM as well as in normal cells increased DNA breaks and HR activity. Furthermore, the inhibition of HDAC8 (by knockdown and OJI-1) inhibited, whereas its overexpression increased genomic instability, as assessed by micronucleus assay, in surviving MM cells. We also demonstrate that HDAC8 interacts with RAD51 and impacts its acetylation. The treatment of MM cells with HDAC8 inhibitor (OJI-1) increased RAD51 acetylation. Next, we examined the in vivo efficacy of the HDAC8 conditional knockdown in a human xenograft mouse model, using H929 cells injected subcutaneously in SCID mice. HDAC8 knockdown not only caused a significant reduction in tumor growth but also increased survival (p=0.0016) compared to mice injected with control cells. Evaluation of tumors from these mice confirmed in vivo inhibition of DNA breaks and HR activity, and induction of apoptosis following HDAC8-knockdown. HDAC8 inhibitor OJI-1 also synergistically increased the cytotoxicity of existing MM drugs including dexamethasone, bortezomib and lenalidomide. In conclusion, our results demonstrate that elevated HDAC8 in MM cells is involved in inhibition of apoptosis but also contributes to increased DNA breaks and dysregulation of homologous recombination and genome stability. Therefore, HDAC8 is a novel target for therapeutic application in MM. Selective and potent HDAC8 inhibitor OJI-1 has shown a favorable therapeutic index with synergistic effect in combination with existing MM drugs. Disclosures Hajek: Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Munshi: Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy.

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Increased Sclerostin Secretion in Multiple Myeloma Results in Stimulation of Osteoclastogenesis and Inhibition of Osteoblastogenesis

Blood ◽

10.1182/blood.v118.21.1819.1819 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1819-1819

Author(s):

Homare Eda ◽

Loredana Santo ◽

Diana D. Cirstea ◽

Samantha Pozzi ◽

Miriam Canavese ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Bone Disease ◽

Research Funding ◽

Plasma Concentrations ◽

Bone Homeostasis ◽

Advisory Committees ◽

Good Target ◽

Osteolytic Bone Disease

Abstract Abstract 1819 Objectives: Osteoblasts (OB) and osteoclasts (OC), are an integral part of the bone microenvironment, and play a crucial role in myeloma growth and survival. Their imbalance results in osteolytic disease and elucidating the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of multiple myeloma (MM). The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis. It is regarded as a good target for osteoporosis treatment, but its role in MM remains to be determined. Our objective was to study the role of sclerostin in MM bone disease and determine if sclerostin directed strategies were a reasonable approach in MM. Methods and Results: Sclerostin concentration in patients' blood plasma and MM cell line supernatant stimulated by IL-6, FGF-2, TNFalpha, BMP7 and TGFbeta was detected by ELISA (ALPCO immunoassays). Increased level of sclerostin was detected in MM patient plasma (n=20, median: 4.73 ng/mL, range: 1.5–19.5 ng/mL). Plasma concentrations were significantly higher (p<0.01) when compared to sclerostin concentration in the plasma of leukemia patients (n=3), gastric cancer patients (n=40) and healthy volunteers (n=4). High sclerostin levels were not associated with extent of bone disease but rather correlated with tumor burden (High B2M, creatinine and LDH, and low Hb) suggesting an autocrine loop for sclerostin production. Because sclerostin is derived from mature OB or orteocytes, we measured levels during OB differentiation but we were unable to detect increased levels. We then measured sclerostin levels in RPMI-8226 MM cell line supernatant either alone or stimulated by cytokines D Systems). Conclusions: These data demonstrate that increased sclerostin levels in MM patients inhibit osteoblastogenesis and stimulate osteoclastogenesis. Taken together, sclerostin may be good target to inhibit myeloma bone disease and help restore normal bone homeostasis. Disclosures: Raje: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.

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A Phase IB, Multi-Center, Open-Label Dose-Escalation Study of Oral Panobinostat (LBH589) and I.V. Bortezomib in Patients with Relapsed Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.3852.3852 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3852-3852 ◽

Cited By ~ 8

Author(s):

Jesús F. San-Miguel ◽

Orhan Sezer ◽

David Siegel ◽

Andreas Guenther ◽

Maria-Victoria Mateos ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Clinical Investigation ◽

Advisory Board ◽

Protease Inhibition ◽

Advisory Committees ◽

Dose Escalation Study ◽

Phase Ib ◽

Synergistic Cytotoxicity

Abstract Abstract 3852 Poster Board III-788 Introduction Panobinostat (LBH589) is a highly potent pan-deacetylase inhibitor (pan-DACi), inclusive of HDAC6, which disrupts aggresome function, promotes accumulation of cytotoxic misfolded protein aggregates and triggers myeloma cell death. Combination of pan-DAC and protease inhibition by co-treatment with panobinostat (PAN) and bortezomib (BTZ) has demonstrated synergistic cytotoxicity in vitro and in vivo in multiple myeloma (MM) cell lines and may provide increased efficacy in patients with MM. The primary objective of this Phase Ib trial is to determine the maximum tolerated dose (MTD) of oral PAN when combined with BTZ in patients with relapsed or refractory MM. Safety, tolerability, PK/PD, and preliminary efficacy are the secondary objectives. Results A total of 29 patients have been enrolled into four completed dosing Cohorts: (I) 10 mg PAN (TIW) + 1 mg/m2 BTZ (i.v., Days 1, 4, 8, 11) during a 21-day cycle; (II) 20 mg PAN + 1 mg/m2 BTZ; (III) 20 mg PAN + 1.3 mg/m2 BTZ; (IV) 30 mg PAN + 1.3 mg/m2 BTZ. Enrollment into Cohort V is ongoing at 25 mg PAN + 1.3 mg/m2 BTZ with 6 patients accrued to date. In Cohorts I– IV, the median number of prior therapies was 3 (range 1–6); 25 patients had at least one prior auto-SCT. Of 16 BTZ pretreated patients, 11 were refractory to their last prior BTZ-based therapy (9 with PD, 2 with SD on BTZ). Median time on study has been 97 days (range 7–424). Overall, the combination of PAN and BTZ was safe and tolerated in Cohorts I - III with one dose-limiting toxicity (DLT) (Gr 4 afebrile neutropenia) in Cohort II. In Cohort IV, four DLTs were reported: two Gr 4 thrombocytopenias,(requiring platelet transfusions), Gr 3 pneumonia, and Gr 3 fatigue. In the subsequent Cohort V, PAN dose was de-escalated. Hematologic adverse events (AEs) have been frequent, including Gr 3/4 thrombocytopenia (25), neutropenia (18), and anemia (6). Non-hematologic AEs included: diarrhea (18), fever (15), nausea (14), fatigue (14), and asthenia (11). A total of 1,778 ECGs were centrally, reviewed with neither QTcF prolongation from baseline >60 msec nor absolute QTcF duration >480 msec noted. Gr 3/4 thrombocytopenia was manageable by dose modification and platelet transfusion; two patients only discontinued for this AE in Cohorts I – III and no hemorrhagic events were reported in association with thrombocytopenia. Encouraging clinical efficacy was observed in all four Cohorts, with 14 responders (partial response [PR] or better) in 28 evaluable patients (50%), including 4 with immunofixation (IF) negative complete response (CR). Four additional patients achieved minor responses, resulting in 64% overall response rate. Responses were also seen in the subset of patients refractory to prior BTZ, suggesting a strong clinical correlate for synergism of the PAN/BTZ combination: 6 of 10 (60%) BTZ-refractory evaluable pts responded, including 4 PR and 2 MR (see Table for details). Dexamethasone (DEX) was introduced at Cycle 2 (or 3) in 9 pts; 11 of 18 pts with a response did not receive DEX, including several pts refractory to BTZ. All 15 patients in Cohorts III and IV treated with the full registered dose of BTZ (1.3 mg/m2) in combination with PAN 20 mg experienced a clinical benefit; however, toxicity in Cohort IV was not acceptable. Conclusion The encouraging clinical anti-myeloma synergism of the PAN and BTZ combination in this trial warrants further clinical investigation in patients with refractory and relapsed MM. Given the frequency of thrombocytopenia and dose adjustments, the dosing schedule in subsequent Phase II/III studies will be modified to take the safety profile and dose-reduction/-interruption pattern into account. Disclosures: San-Miguel: Novartis: Advisory Board, Consultancy, Honoraria; J&J: Advisory Board, Consultancy, Honoraria; Millenium: Advisory Board, Consultancy, Honoraria; Celgene: Advisory Board, Consultancy, Honoraria. Sezer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Siegel:Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Speakers Bureau. Guenther:Novartis: Consultancy, Research Funding. Mateos:Ortho Biotech: Speakers Bureau; Novartis: Honoraria. Cavo:Novartis: Honoraria. Blade:Novartis: Honoraria; Janssen-Cilag: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Boccadoro:Celgene: Honoraria; Janssen Cilag: Honoraria. Bengoudifa:Novartis Pharma AG: Employment. Klebsattel:Novartis Pharma AG: Employment. Bourquelot:Novartis Pharma AG: Employment. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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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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Disruption of DEPTOR/mTORC1/mTORC2 Signaling Cascade Using a Novel Selective mTOR Kinase Inhibitor AZD8055 Results In Growth Arrest and Apoptosis In Multiple Myeloma Cells

Blood ◽

10.1182/blood.v116.21.791.791 ◽

2010 ◽

Vol 116 (21) ◽

pp. 791-791 ◽

Cited By ~ 1

Author(s):

Diana Cirstea ◽

Teru Hideshima ◽

Loredana Santo ◽

Samantha Pozzi ◽

Sonia Vallet ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Drug Resistance ◽

Cell Growth ◽

Cell Survival ◽

Board Of Directors ◽

Research Funding ◽

Kinase Inhibitor ◽

Advisory Committees ◽

Mtor Kinase

Abstract Abstract 791 Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM. Disclosures: Guichard: AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

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TAS-117, a Novel Selective Akt Inhibitor Demonstrates Significant Growth Inhibition in Multiple Myeloma Cells in Vitro and in Vivo

Blood ◽

10.1182/blood.v120.21.942.942 ◽

2012 ◽

Vol 120 (21) ◽

pp. 942-942 ◽

Cited By ~ 1

Author(s):

Naoya Mimura ◽

Hiroto Ohguchi ◽

Diana Cirstea ◽

Francesca Cottini ◽

Gullu Topal Gorgun ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Akt Inhibitor ◽

Advisory Committees ◽

Significant Growth Inhibition

Abstract Abstract 942 The PI3K/Akt pathway mediates multiple myeloma (MM) cell growth and drug resistance, and targeting this molecule is a promising therapeutic option. In this study, we examined anti-MM activities of TAS-117 (TAIHO PHARMACEUTICAL CO., LTD., JAPAN), a selective potent Akt inhibitor in MM cell lines including MM.1S, MM.1R, OPM1 and H929 cells with high level of baseline Akt phosphorylation. TAS-117 induced significant growth inhibition in these cell lines, associated with downregulation of phosphorylation (Ser473 and Thr308) of Akt and downstream molecule FKHR/FKHRL1, without cytotoxicity in normal peripheral blood mononuclear cells. TAS-117 triggered G0/G1 arrest followed by apoptosis, evidenced by increased annexin V-positive cells, in both MM.1S and H929 cell lines. Apoptosis was further confirmed by cleavage of caspase-8, -3 and PARP. Interestingly, TAS-117 also induced: autophagy, evidenced by increased LC3-II; as well as endoplasmic reticulum (ER) stress, confirmed by induction of phospho-eIF2α, phospho-IRE1α and a molecular chaperone BiP/GRP78. Since the bone marrow (BM) microenvironment plays a crucial role in MM cell pathogenesis including drug resistance, we further examined the effect of TAS-117 in the presence of BM stromal cells (BMSCs). TAS-117 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt. Importantly, TAS-117 inhibited secretion of IL-6 from BMSCs, and exogenous IL-6 and IGF-1 did not block cytotoxicity induced by this agent. We have previously shown the bortezomib activates Akt, and that Akt inhibition with bortezomib triggers synergistic MM cell cytotoxicity. TAS-117 enhanced bortezomib-induced cytotoxicity in MM.1S cells, associated with increased CHOP followed by PARP cleavage, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. TAS-117 also enhanced cytotoxicity induced by other therapeutic agents (ie, rapamycin, dexamethasone, 17-AAG) in MM.1S cells. Finally, we examined anti-MM activities of TAS-117 in a xenograft murine model. Oral administration of TAS-117 for 14 days significantly inhibited growth of H929 plasmacytoma and was well tolerated. Taken together, the novel and selective Akt inhibitor TAS-117 blocks MM cell growth in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Shimomura: TAIHO PHARMACEUTICAL CO., LTD.: Employment. Utsugi:TAIHO PHARMACEUTICAL CO., LTD.: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

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Rational Combination Treatment of a Novel Selective mTOR Kinase Inhibitor AZD8055 with IGF-1R Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.4023.4023 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4023-4023

Author(s):

Diana Cirstea ◽

Teru Hideshima ◽

Loredana Santo ◽

Homare Eda ◽

Tyler A. Scullen ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Combination Treatment ◽

Research Funding ◽

Akt Signaling ◽

Advisory Committees ◽

Kinase Inhibition ◽

Akt Activation ◽

Mtor Kinase

Abstract Abstract 4023 Background: mTOR kinase-targeted therapy is in the early phase of clinical evaluation in multiple myeloma (MM). Despite promising preclinical results with mTOR inhibitors, resistance to this class of drugs in MM patients may occur due to feedback Akt activation by mTORC1. This led to the development of mTORC1/2 inhibition strategies in the treatment for MM, predicated upon the rationale that mTORC2 inhibitors prevent inhibition of mTORC1 blockade-induced feedback AKT activation by mTORC1 inhibitors. Indeed, our previous studies using a novel dual mTORC1 and mTORC2 selective inhibitor AZD8055 show MM cell growth inhibition via apoptosis, associated with inhibition of mTORC1 and mTORC2 signaling, including rapamycin-resistant 4E-BP1 (downstream of mTORC1) and Akt as well as NDRG1 (an effector of mTORC2). Importantly, AZD8055 also inhibited PI3K/Akt signaling and related MM cell growth induced by cytokines (i.e., IL-6, IGF-1) or co-culture with bone marrow stromal cells (BMSCs). Recent studies, however, reveal that constitutively activated Akt signaling negatively regulates IGF-1 receptor (IGF-1R) at the transcriptional level, independent of mTOR activity. Moreover, AKT-induced IGF-1R down-regulation reduces sensitivity of IRS1 to IGF-1 stimulation. We have also shown that IGF-1R inhibitor triggers significant MM cell toxicity. Methods and Results: In this study, we therefore hypothesized that mTORC2 blockade may upregulate IGF-1R expression and/or activity via Akt modulation in MM cells, and that IGF-1R blockade may enhance the cytotoxic effects of mTOR kinase inhibition in MM cells. We first examined the tyrosine phosphorylation sites (Y1135/1136) in the activation loop of the IGF-1R kinase domain in three MM cell lines (MM.1S, OPM1 and RPMI8226) treated with AZD8055 or rapamycin. AZD8055 induced more pronounced upregulation of p-IGF-1R in MM.1S and OPM1 MM cells than rapamycin at earlier time periods. IGF-1 clearly upregulated Akt phosphorylation in MM cells; however, it had no effect on mTOR phosphorylation (Ser2481). Moreover, AZD8055-treated cells exposed to IGF-1 sustained p-Akt (Ser473) expression, while p-mTOR (Ser2481) remained fully inhibited. These results suggest that IGF-1/IGF-1R signaling may bypass mTORC2/Akt when mediating p-Akt (Ser473) upregulation. Moreover, reactivation of IGF-1 signaling in MM cells in the context of mTOR kinase inhibitors suggests that MM may survive in an IGF-1 R–dependent fashion. We therefore next treated MM.1S, OPM1 and RPMI8226 cells with AZD8055, in the presence or absence of IGF-1. MM.1S and OPM1 MM cells (with higher Akt baseline activity) partially escaped AZD8055 cytotoxicity, while RPMI8226 MM cells (with lower Akt activity) did not. Moreover, the addition of blocking IGF-1R antibody or of IGF-1R inhibitor enhanced AZD8055 cytotoxicity in MM.1S and OPM1 cells. Conclusions: Our study therefore shows interaction of mTOR/Akt and IGF-1R/Akt pathways in MM tumors with IGF-1-enabled Akt activation. Importantly, they suggest that combination treatment with AZD8055 and IGF-1R inhibitor is a promising strategy to mTOR kinase inhibition in MM with potential IGF-1R/Akt signaling mediated survival. Disclosures: Hideshima: Acetylon Pharmaceuticals, Inc.: Consultancy. Guichard:AstraZeneca: Employment, Shareholder Other. Anderson:Onyx: 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. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

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Target Expression, Preclinical Activity and Mechanism of Action of EM801: A Novel First-in-Class Bcma T-Cell Bispecific Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.117.117 ◽

2015 ◽

Vol 126 (23) ◽

pp. 117-117 ◽

Cited By ~ 8

Author(s):

Anja Seckinger ◽

Jose Antonio Delgado ◽

Laura Moreno ◽

Brigitte Neuber ◽

Anna Grab ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Board Of Directors ◽

T Cell Activation ◽

Mechanism Of Action ◽

Research Funding ◽

Advisory Committees

Abstract Background. T-cell bispecific antibodies (TCBs) simultaneously binding CD3 on T-cells and individual tumor antigens, activate T-cells and destroy tumor antigen carrying cells. B-cell maturation antigen (BCMA), a surface antigen reported to be expressed on normal and malignant plasma cells (PCs), could represent a potentially promising target for TCBs in multiple myeloma (MM). The Aim of our study was to: i) assess expression of BCMA in normal and malignant PCs as well as cells of the bone marrow (BM) microenvironment by gene expression profiling and flow cytometry to validate it as potential clinical target for TCBs; ii) to evaluate activity of EM801 as member of a novel class of BCMA-TCBs in vitro on primary myeloma cells and in vivo in the H929-xenograft reconstituted NOG mouse model; and iii) to delineate its mechanism of action. Results. Expression. We investigated the expression of BCMA in CD138-purified PCs from BM aspirates obtained from 726 patients including MGUS (n=62), asymptomatic (n=59) and symptomatic MM (605), as well as different BM cellular subsets from healthy donors (n=10 PCs; plasmablasts, memory B-cells, T-cells, CD34+, CD14+, CD15+, n=5 each; n=8 mesenchymal stromal cells) using Affymetrix DNA-microarrays. BCMA expression was observed in malignant PC from 723/726 (99.5%) MGUS and MM patients, 10/10 normal PCs and 5/5 plasmablasts; gene expression of BCMA was undetectable in all other normal BM subsets. Using multiparameter flow cytometry, BCMA surface expression on malignant PCs was confirmed in 40/40 patients while being absent on normal BM cells. BCMA is thus a potential target in virtually all myeloma patients. Activity. In vitro, EM801 induced concentration dependent significant cell death in malignant plasma cells in BM-samples of 21/28 (75%) previously untreated and 8/10 (80%) relapsed/refractory MM patients in concentrations ranging from 10pM to 30nM. No or only minor unspecific toxicity on cells of the BM microenvironment was observed. In vivo efficacy of EM801 was studied in a subcutaneous H929 myeloma cell line xenograft model in NOG (NOD/Shi-scid/IL-2Rγnull) mice reconstituted with human PBMCs. Three doses of EM801, i.e. 0.026, 0.26 and 2.6 nM/kg, the same doses of a BCMAxCD3-(scFv)2 and two control groups were investigated (n=9 mice/group). Three weekly intravenous doses were given, starting on day 19 after tumor cell injection when tumor volumes were 293±135 mm3. On day 47, all mice from control groups had their tumors grown beyond 2000 mm3 and were euthanized for ethical reasons. In contrast, at 2.6 nM/kg (0.5 mg/kg) EM801 tumor regression was already observed after the second i.v. injection in 6/9 animals and the tumor regressed to 16±3 mm3 on day 47. BCMAxCD3-(scFv)2 bispecific antibody without Fc did not show any efficacy at all doses studied. Regarding the mechanism of action, we first demonstrated that EM801 effectively binds myeloma cells and T-cells with a strength of 1622±410 pN (5-10 fold of control) as measured by atomic force microscopy. Secondly, increasing concentrations (0.03-30nM) of EM801 led to progressive T-cell activation in primary BM samples, with significantly increased levels of CD69 (P<0.001), CD25 (P<0.001) and HLADR (P=0.001) expression in both CD4 and CD8 T-cells as compared to an unspecific TCB. Thirdly, EM801 induced significant secretion of interferon-γ (19-3000 pg/ml), granzyme B (68-2986 pg/ml), and perforin (145-3712 pg/ml) as measured by ELISA, together explaining the strong in vitro and in vivo activity of EM801. Conclusions. BCMA is selectively expressed at the RNA (723/726) and protein (40/40) levels on malignant PCs from virtually all MM patients, and thus represents a promising TCB-target. The novel BCMA-TCB EM801 was effective in vitro in 29/38 (76%) primary MM patients' BM samples at picomolar to low nanomolar concentrations, easily achievable in vivo in patients, as well as in the H929-xenograft reconstituted NOG mouse model at 0.5 mg/kg once a week. Neither in vitro (the BM microenvironment) nor in vivo the compound shows significant toxicity or side effects. EM801 confers cytotoxicity by effectively coupling T-cells with malignant PCs, inducing T-cell activation, secretion of interferon-γ, granzyme B and perforin, and thereby effectively killing malignant PCs. EM801 is thus a promising new compound for the treatment of multiple myeloma to be investigated in clinical phase I/II trials. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Neuber:EngMab AG: Research Funding. Vu:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Strein:BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees; EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hundemer:EngMab AG: Research Funding. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria. Hose:Takeda: Other: Travel grant; EngMab AG: Research Funding. Paiva:Celgene: Consultancy; Janssen: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Sanofi: Consultancy.

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Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Blood ◽

10.1182/blood-2018-99-111337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4481-4481

Author(s):

Denise Toscani ◽

Martina Chiu ◽

Giuseppe Taurino ◽

Emanuela Vicario ◽

Valentina Marchica ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Bone Disease ◽

Research Funding ◽

Bone Cells ◽

Bone Microenvironment ◽

Advisory Committees ◽

Osteolytic Lesions ◽

Osteolytic Bone Disease ◽

The Relationship

Abstract Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions. Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions. We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression. Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested. Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. Disclosures Aversa: Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

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Inhibition of Sialylation Impairs Adhesion on Madcam-1 and E-Selectin and Sensitize Multiple Myeloma Cells to Bortezomib in a Xenograft Mouse Model

Blood ◽

10.1182/blood-2018-99-118361 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3204-3204

Author(s):

Alessandro Natoni ◽

Mariah Farrell ◽

Heather Fairfield ◽

Lucy Kirkham-McCarthy ◽

Matt Macauley ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Board Of Directors ◽

Research Funding ◽

Immune Cell ◽

Sialyl Lewis X ◽

Advisory Committees ◽

Selectin Ligands

Abstract Introduction Multiple myeloma (MM) is a cancer of clonal plasma cells that hijack the bone marrow (BM) niche to create a drug resistant, incurable malignancy. Aberrant sialylation has been linked to immune cell evasion, drug resistance, and metastasis in cancer; indeed sialyltransferases, including ST3GAL1, ST3GAL4 and ST3GAL6, are aberrantly expressed in many cancers (Glavey et al., 2014). We have previously shown that targeting ST3GAL6 in MM cells inhibits their ability to extravasate and colonize the BM in mouse models (Glavey et al., 2014). Moreover, we also showed that a subpopulation of MM cells expresses functional E-Selectin ligands which, upon expansion, gives rise to a more aggressive disease and resistance to bortezomib in mice (Natoni et al., 2017). Based off these findings, we herein investigated whether inhibiting sialylation in E-selectin-enriched MM cells with 3Fax-Neu5Ac, a small molecule sialyltransferase inhibitor, could alter the ability of these cells to home in the BM and restore bortezomib sensitivity in vivo. We hypothesized that inhibiting homing of MM cells to the BM will improve survival and that co-treatment with bortezomib and 3Fax-Neu5Ac will have a synergistic effect. Methods E-selectin ligands enriched MM1S cells (either positive or negative for GFP/Luciferase) were derived from parental cells by cell sorting using the HECA-452 antibody, which recognize sialofucosylated E-selectin ligands. We then determined the 3Fax-Neu5Ac dose and exposure times needed to decrease sialylation on these MM cells without causing toxicity. HECA-452-enriched MM1S cells were pretreated with 3Fax-Neu5Ac or vehicle for 7 days before being injected into SCID-beige mice and then treated with vehicle or bortezomib (0.3 mg/kg twice a week). Mice were analyzed via bioluminescence imaging (BLI) to monitor tumor progression and weighed twice a week. Mice were euthanized when they began to show paralysis under our IACUC protocol. 3Fax-Neu5Ac pretreated HECA-452 MM1S cells were also tested in vitro for their ability to adhere and roll on VCAM-1, MAdCAM-1 and E-Selectin under shear stress and to respond to bortezomib in co-culture with HS5 cells. Results Treatment of HECA-452 MM1S cells with 3Fax-Neu5Ac, at 300 μM for 7 days significantly reduced sialylation on these cells. Importantly, reducing sialylation with 3Fax-Neu5AC reduced tumor burden and increased survival, although this did not reach significance for survival (Figure 1A). Both vehicle- and 3Fax-Neu5Ac-treated cells significantly responded to bortezomib in the first 5 weeks of the in vivo study (Figure 1B). However, the HECA-452 MM1S cells did not show increased survival when treated with bortezomib suggesting an acquired mechanism of resistance in vivo. Importantly, pretreatment of the HECA-452 MM1S with 3Fax-Neu5Ac could improve survival of these mice preventing bortezomib resistance. In vitro, the HS5 stromal cells protected the HECA-452 MM1S cells from bortezomib and pretreatment with 3Fax-Neu5Ac partially reverted this protection. Moreover, the HECA-452 MM1S cells pretreated with 3Fax-Neu5Ac displayed reduced adhesion on MAdCAM-1 and E-selectin. Conclusions Sialylation plays an instrumental role in bone homing, BM colonization, and drug resistance of MM cells. Pretreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac decreased their sialylation, restored sensitivity to bortezomib in vivo and prolonged survival in mice. This is likely because 3Fax-Neu5Ac pretreatment has multiple effects on MM cells including reducing cell adhesion mediated-drug resistance and adhesion to key molecules involved in BM homing such as MAdCAM-1 and E-selectin. The reduced adhesion on E-selectin is most likely due to the disruption of E-selectin ligands on the surface of MM cells as they require Sialyl Lewis X to function. Notably, we also found that de-sialylation impairs adhesion on MAdCAM-1 (3Fax-Neu5Ac vs DMSO P=0.038) which, together with E-selectin, is another critical BM homing receptor. This data suggests for the first time that sialylation may controls the affinity of integrin α4β7 and its counter-receptor MAdCAM-1. In turn, this would reduce BM homing and increase MM cells in the circulation were they are more prone to the cytotoxic effects of bortezomib. This study supports the importance of targeting sialylation in MM and provides a strong rationale for further clinical translation of this novel approach. Disclosures O'Dwyer: Glycomimetics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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