Xenograft Model Of Human Multiple Myeloma Research Articles

Discovery of a Novel, First-in-Class Bfl-1 BH3 Mimetic with Pro-Apoptotic Activity

Background: Intrinsic apoptosis is regulated by the B Cell lymphoma 2 (BCL-2) protein family. Anti-apoptotic family members, such as BCL-2, MCL-1 and BFL-1, sequester their pro-apoptotic counterparts through a highly conserved BH3 binding groove. Disruption of this balance leads to mitochondrial pore formation and subsequent cell death. To evade apoptosis, tumors often upregulate the expression of one or more anti-apoptotic family members and as such they have become attractive targets for anti-cancer drug development. BFL-1 (‘Bcl-2 related gene expressed in fetal liver’, gene name BCL2A1) is a lesser-known anti-apoptotic family member, physiologically mainly expressed in the hematopoietic system. BCL2A1 overexpression has been reported in many types of B cell lymphoma. Diffuse Large B Cell Lymphoma (DLBCL) patient sample analysis showed that BCL2A1 expression levels are second only to MCL1 within the anti-apoptotic family. Expression of BFL-1 was shown to be upregulated in MYC+/BCL2+ double hit lymphoma cell lines treated with the BCL-2 inhibitor, Venetoclax, in vivo. Interestingly, BFL-1 positive lymphomas are less sensitive to inhibition of BCL-2 and MCL-1. Hence, targeting BFL-1 is a rational and attractive strategy in DLBCL to improve clinical outcome. Methods: Using hit identification screens followed by iterative structure-activity relationship (SAR) analyses and structure-based design, compound A, a first-in-class, selective BFL-1 BH3 mimetic, was identified. Compound A was evaluated using biochemical, cellular in vitro and in vivo tumor efficacy models. Results: Compound A is a highly potent and selective compound in biochemical assays. In BFL-1 sensitive lymphoma cell lines characterized by high BFL-1 protein expression, compound A was shown to displace BAK and BIM from BFL-1, leading to downstream cleavage of caspase 3 and cell death. Interestingly, these cellular models were resistant to Venetoclax. Compound A exhibited tumor growth inhibition in an engineered BFL-1 overexpressing, MCL-1 KO human multiple myeloma xenograft model. However, in the high BFL-1 expressing SU-DHL-2 DLBCL human xenograft model, compound A did not induce significant PD (cleaved caspase 3) and efficacy in vivo. Conclusion: First-in-class selective BFL-1 BH3 mimetics are shown to specifically inhibit cell growth of lymphoma cell lines in vitro and can induce efficacy in vivo in a BFL-1 overexpressing model. Further studies may be useful to determine whether compound A elicits anti-tumor efficacy in lymphoma alone or in combination with other anti-apoptotic agents.

Immunotherapeutic Development of a Tri-Specific NK Cell Engager Recognizing BCMA

Chemotherapy-refractive multiple myeloma (MM) is serious and life-threatening, and better treatments are urgently needed. BCMA is a prominent marker on the cell surface of MM cells, rendering it an accepted target for antibody therapy. Considering that MM is a liquid tumor and immunotherapy has enjoyed success against leukemia, we devise an approach designed to enhance NK cell activity against MM. Ordinarily, NK cells function to naturally survey the body and eliminate malignant cells. Our platform approach is designed to enhance NK function. A tri-specific immune-engaging TriKE is manufactured, consisting of a camelid nanobody VHH antibody fragment recognizing CD16 expressed on NK cells and an scFv antibody fragment specifically recognizing BCMA. These two fragments are crosslinked by the human cytokine interleukin-15 (IL-15) known to have prominent activating effects on NK cells. The molecule, when tested by flow cytometry, shows activation of NK cells in their numbers and activity. Additionally, the molecule demonstrates anti-cancer effects in an in vivo xenograft model of human MM. We believe that the drug will have the capability of enhancing NK cells at the site of the immune synapse, i.e., the effector:target cell interface, and this will promote cancer remissions.

Expanded natural killer cells potentiate the antimyeloma activity of daratumumab, lenalidomide, and dexamethasone in a myeloma xenograft model

The development of new treatment agents in recent decades has significantly improved the survival of patients with multiple myeloma (MM). Nonetheless, MM remains an incurable disease; therefore, novel combination therapies are required. Natural killer (NK) cells are one of the safest immunotherapeutic options. In this study, we found that the anti-myeloma activity of expanded NK cells (eNKs) was improved by daratumumab, lenalidomide, and dexamethasone (DRd) in an MM xenograft mouse model. NK cells expanded from peripheral blood mononuclear cells collected from MM patients were highly cytotoxic against DRd pretreated tumor cells in vitro. To mimic the clinical protocol, a human MM xenograft model was developed using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. MM bearing mice were randomly divided into six groups: no treatment, eNK, Rd, Rd + eNKs, DRd, and DRd + eNKs. DRd significantly enhanced the cytotoxicity of eNKs by upregulating NK cell activation ligands and effector function. DRd in combination with eNKs significantly reduced the serum M-protein level and prolonged mouse survival. In addition, DRd significantly increased the persistence of eNK and homing to MM sites. These results show that the anti-myeloma activity of ex vivo-expanded and activated NK cells is augmented by the immunomodulatory effect of DRd in MM-bearing mice, suggesting the therapeutic potential of this combination for MM patients.

Anti-Myeloma Effect of Compound K and Rh2 in a Multiple Myeloma Xenograft Model

Introduction: Several ginsenosides have demonstrated anti-cancer activity due to their ability to inhibit DNA synthesis and angiopoiesis, inducing cancer death. Rh2, a PPD-type 20(S)-ginsenoside, has been extensively studied as a major ingredient with antineoplastic properties. Moreover, the characteristics of compound K (CK), a PPD ginsenoside metabolite, resulting from the metabolic activity of enterobacteria, have been investigated. The effect of ginsenoside has been actively studied in leukemia but not in multiple myeloma (MM). Thus, the aim of study was to identify ginsenosides that have toxic effects against MM cells and to elucidate the underlying molecular mechanism. Methods: First, we screened the toxic effects of ginsenosides Rb1, Rb3, Rg1, Rg3, Rh1, Rh2, and CK at a concentration of 50 μM in several MM cell lines (ARH77, U266, and RPMI8226) after 24 hours of exposure. Furthermore, ARH77 cells were incubated with Rh2 and CK at 0, 20 μM, 50 μM, 60 μM, and 70 μM for 24 hours, and the DNA content at each stage of the cell cycle was detected. The potential cytotoxic effect of Rh2 and CK was investigated in normal hematopoietic stem cells (HSCs) isolated from healthy donors using primary peripheral blood stem cells (PBSCs). For this experiment, HSCs were isolated using a PBSC collection procedure after the recruitment of healthy donors. Briefly, granulocyte colony-stimulating factor was administered continuously until the white blood cell count exceeded the threshold of 1 × 109 cells/L. The subjects underwent leukapheresis once or twice using a blood cell separator. To quantify the production of PBSCs from each leukapheresis, flow cytometry was carried out to quantify CD34+ cells. To establish a human MM xenograft model, RPMI8226-RFP-FLuc cells (5 × 106 per mouse) were intravenously injected into 9-12-week-old male and female NOD/SCID IL-2Rγnull (NSG) mice. To assess the anti-MM effect of CK and Rh2 in the MM xenograft model, NSG mice were divided into the following treatment groups: Experiment set 1: no treatment (PBS control), CK (15 mg/kg/day), Rh2 (15 mg/kg/day), K (30 mg/kg/day), and Rh2 (30 mg/kg/day). Mice were treated with either CK or Rh2 (15 or 30 mg/kg/day) by intraperitoneal injection for 3 days in a week at a 1-day interval. Experiment set 2: no treatment (PBS control), CK (7 mg/kg/day), Rh2 (7 mg/kg/day), and Lenalidomide (1 mg/kg/day). Mice were treated with either CK or Rh2 (7 mg/kg/day) by intravenous injection for 3 days in a week at a 1-day interval. Lenalidomide (1 mg/kg/day) treatment was given by oral administration from day 14 for 5 consecutive days in a week, and it was repeated for 3 weeks at a 2-day interval in each week. Tumor growth was monitored weekly by bioluminescence imaging in the dorsal view; 10 min before imaging, mice were intraperitoneally injected with D-Luciferin (150 mg/kg/mouse). Results: The ginsenosides Rh2 and CK inhibited the cell growth, exerted cytotoxicity, and induced G1 phase cell cycle arrest in human MM cells. However, these treatments demonstrated no cytotoxic effect on the self-renewal and the differentiation capacity of normal primary PBSCs. This observation implied that the cytotoxic activity of Rh2 and CK might selectively target MM cells and that these ginsenosides are safe in other cell types. Finally, we studied the anti-MM effect of CK and Rh2 in a clinically relevant human MM xenograft model. PBS was given in the no treatment control group and all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytoma, which led to death within 7 weeks. Notably, mice treated with CK and Rh2 showed significant inhibition of tumor growth and a longer survival. Conclusions: Ginsenosides CK and Rh2 inhibited the growth of MM cells in an in vitro and in vivo model without any significant cytotoxic effect on the self-renewal and differentiation capacity of PBSCs. Thus, these ginsenosides could be used in MM treatment and can be combined with drugs without exerting any severe toxic effects. Keywords: Ginsenosides; Rh2; Compound K; Multiple myeloma Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Repotrectinib's Anti-MM Effects in Combination with Bortezomib, Carfilzomib, Cyclophosphamide, and Dexamethasone in Vitro and Ex Vivo

Introduction: The proteasome inhibitors bortezomib and carfilzomib are anticancer drugs which are indicated for the treatment of multiple myeloma (MM). The alkylating agent cyclophosphamide is also an effective drug against MM. Glucocorticoids including dexamethasone induce MM cell apoptosis and are effective in MM. However, the disease remains incurable with currently available therapies. Repotrectinib (Turning Point Therapeutics, San Diego, CA) is a novel next-generation ROS1/TRK/ALK-tyrosine kinase inhibitor (TKI). Repotrectinib also inhibits the Janus kinase (JAK) 2 and indirectly down-regulates STAT5 phosphorylation in the SET2 cancer cell line harboring a JAK2 V617F tyrosine kinase mutation (Drilon et al 2018). We have previously shown the preclinical anti-MM effects of JAK1/2 inhibitors in combination with corticosteroids and other anti-MM drugs in vitro and in vivo. Although activation of JAK2 and PI3K signaling is often detected in hematological malignancies, there is no data regarding the potential anti-MM effects of repotrectinib alone or in combination with other anti-MM drugs. Thus, we evaluated the anti-myeloma effects of repotrectinib alone and in combination with bortezomib, carfilzomib, cyclophosphamide, and dexamethasone in vitro using MM cell lines and ex vivo using the human MM xenograft model LAG- λ1. Methods: The human MM cell lines U266, RPMI8226 and MM1S were obtained from ATCC. MCs were isolated from MM patients' BM aspirates. Cell viability was quantified using the MTS assay. For the ex vivo studies, cells were isolated from the MM xenograft LAG-λ1 after it was established in SCID mice. We determined the IC curves using the MM cell lines and fresh BMMCs from 6 MM patients. Next, we combined repotrectinib with other active anti-MM drugs (carfilzomib, bortezomib, cyclophosphamide, and dexamethasone) using these same three MM cell lines and BMMCs from 6 MM patients to evaluate the anti-proliferative effects of these combinations. Results: We first evaluated the anti-MM effects of repotrectinib in combination with bortezomib or carfilzomib by using the MM1S, RPMI-8226, and U266 MM cell lines. Specifically, the results with repotrectinib in combination with carfilzomib showed enhanced anti-MM effects when compared to repotrectinib alone or carfilzomib alone. We also determined the anti-MM effects of repotrectinib alone and in combination with carfilzomib by using isolated LAG- λ1 MM xenograft cells. Repotrectinib alone inhibited tumor cell proliferation in a concentration dependent manner (IC50 4.5 µM) and in combination with carfilzomib synergistically inhibited LAG- λ1 MM tumor cell proliferation. These results were not seen when it was combined with bortezomib. We studied BMMCs from 3 PD and 3 CR patients using a cell proliferation assay. The results showed that repotrectinib alone minimally inhibited MM tumor cell proliferation among patients with PD. Repotrectinib combined with carfilzomib showed enhanced anti-proliferative effects compared with either drug alone among BMMCs from PD patients but not those in CR. In contrast, bortezomib did not show this enhanced anti-proliferative effect. We also determined the anti-MM effects of repotrectinib in combination with cyclophosphamide using ex vivo and in vivo studies. The results showed that repotrectinib in combination with cyclophosphamide enhanced the inhibition of LAG- λ1 MM tumor cell and fresh MM BMMC proliferation compared with repotrectinib or cyclophosphamide alone. We further determined the anti-MM effects of repotrectinib in combination with dexamethasone. The results showed that repotrectinib in combination with dexamethasone reduced LAG- λ1 MM cell proliferation in a concentration dependent manner and enhanced inhibition in 2 PD patients' samples but not the other PD patient or 3 CR patients' samples. Summary Single agent repotrectinib decreased MM tumor cell proliferation in all 3 MM cell lines and reduced LAG- λ1 tumor cell proliferation ex vivo. Repotrectinib also reduced proliferation of BMMCs from MM patients with PD but not those in CR. Repotrectinib in combination with carfilzomib, cyclophosphamide or dexamethasone showed enhanced anti-proliferative effects compared with the drugs alone. The results suggest that this kinase inhibitor may have activity in treating MM patients in combination with other active anti-MM agents. Further evaluation is warranted.

Anti-Myeloma Effects of Panobinostat in Combination with Duvelisib

Introduction: Panobinostat (Secura Bio) is a non-selective histone deacetylase inhibitor (HDACi) that has shown anti-tumor activity in preclinical studies in both solid and hematological malignancies. Duvelisib (Secura Bio) is an FDA-approved oral drug and a potent small molecule inhibitor of the delta (δ) and gamma (γ) isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. There is no data regarding in combination of an HDACi with duvelisib for multiple myeloma (MM) treatment. We evaluated the anti-myeloma effects of the panobinostat in combination with duvelisib.Methods: The human MM cell lines U266, RPMI 8226 and MM1s were obtained from ATCC. Primary MM tumor cells were isolated from MM patients' bone marrow (BM) aspirates and mononuclear cells (MCs) were isolated. The cells were seeded at 10 5 cells/well in 96-well plates and incubated for 24 h in the presence of vehicle, panobinostat or duvelisib alone, or the two drugs together for 48 h. Cell viability was quantified using the MTS cell proliferation assay. For the apoptosis assay, U266, RPMI 8226, MM1s, or primary MM tumor cells were cultured at 1 x 10 6 cells per well for 48 hours at 37°C in 5% CO2 with or without the addition of panobinostat or duvelisib. Next, the cells were washed twice with PBS, re-suspended in binding buffer, and stained with FITC-conjugated annexin V and fluorescent dye propidium iodide (PrI) following annexin V assay standard protocol (BioVision, USA). For each drug treatment, 1 x 10 5 gated events were recorded.Results and Discussion: The MTS cell proliferation assay showed that panobinostat alone inhibited cell proliferation of the MM cell lines U266 and MM1s but not RPMI8226. Panobinostat alone induced concentration-dependent inhibition of BMMCs. Panobinostat (20nM) in combination with duvelisib markedly increased inhibition of primary MM cell proliferation. Panobinostat alone induced MM cell apoptosis in all three MM cell lines. Panobinostat in combination with duvelisib enhanced MM cell apoptosis in MM1s but not U266 and RPMI 8226 cells. We further examined apoptosis of primary MM cell treated with panobinostat alone and in combination with duvelisib. The results showed panobinostat alone induced primary MM cell apoptosis and in combination with duvelisib markedly increased the induction of apoptosis. This study illustrates that the combination of panobinostat and duvelisib shows potent anti-MM effects in vitro and we are currently evaluating the anti-MM effects of this combination in vivo using our human MM xenograft models.*The study was partially funded by Secura Bio, Inc DisclosuresNo relevant conflicts of interest to declare.

Preclinical Assessment of CDR101 - a BCMAxCD3xPD-L1 Trispecific Antibody with Superior Anti-Tumor Efficacy

BCMAxCD3 targeting therapies have demonstrated anti-myeloma activity, and high minimal residual disease negativity rates can be achieved with this approach in heavily pre-treated patients with relapsed or refractory multiple myeloma (RRMM). Despite these promising clinical results, patients eventually develop resistant disease and relapse. Thus, there is a high need for novel BCMA therapies that can evade the resistance mechanisms and provide more durable responses. Recently, we reported on the promising activity of the Local Activator and T cell Engager (LocATE) technology, a trispecific molecule that targets CD3, BCMA and PD-L1, redirecting T cells to multiple myeloma (MM) cells while selectively counteracting PD-L1/PD-1 induced immunosuppression at the immune synapse (ASH, 2020). Here we present CDR101, an optimized LocATE candidate with potential for clinical development.First, we analyzed the ability of CDR101 to induce PBMC-mediated cytotoxicity in two MM cell-lines expressing BCMA (U-266 and NCI-H929) and compared it to four BCMAxCD3 bispecific formats currently in clinical development (a half-life extended BCMAxCD3 BiTE, a BCMA-TCB, and two different BCMAxCD3 bispecific monoclonal antibodies) alone or in combination with a separate PD-L1 blocking antibody. CDR101 resulted in at least 10-fold increased T cell-mediated target cell lysis compared to control BCMAxCD3 bispecifics. Strikingly, CDR101 also resulted in increased MM cell killing when compared to free, independent combinations of BCMAxCD3 bispecifics and the PD-L1 inhibitor. These results, together with the observation that MM cells upregulate the expression of PD-L1 in response to treatment with BCMAxCD3 bispecifics, suggest that the superior effect of CDR101 could be attributed to preferential and highly selective inhibition of the PD-1/PD-L1 axis at the cellular interaction within the immune synapse.Next, bone marrow aspirates from newly diagnosed and RRMM patients were treated with increasing concentrations of CDR101 or a BCMAxCD3 bispecific control. After 24h of incubation, percentage of viable CD138-positive cells and activation status of autologous T cells were analyzed by FACS. Overall, CDR101 potently induced lysis of primary MM cells independently of the E:T ratio (range of E:T ratio between 1.3:1 and 33:1). CDR101 achieved higher target cell killing in all samples compared to the bispecific control, with at least 2-fold difference in 3 out of 4 samples at the highest concentration tested. Concomitantly, CDR101 induced a dose-dependent increase of the T cell activation marker CD25, corroborating the ability of CDR101 to counteract PD-L1/PD-1 induced immunosuppression.In vivo anti-tumor activity of CDR101 was evaluated using a human MM (NCI-H929) xenograft model in NPG mice. Treatment with four different doses of CDR101 or BCMAxCD3 bispecific control demonstrated that CDR101 induced stronger and more durable responses compared to the bispecific control leading to complete tumor regression in 55 out of 60 mice at the last day of treatment (day 29) with no relapse until the end of the observation time (day 41).Collectively, CDR101 demonstrated that targeting BCMA with simultaneous blockade of PD-L1 leads to improved myeloma cell killing compared to clinically validated therapies. In contrast to high-affinity PD-L1 immune checkpoint inhibitors, CDR101 selectively inhibits PD-L1 at the immune synapse preventing on-target off-tumor effects. This is expected to translate into a decreased incidence of immune related adverse events (irAEs) and better efficacy arguing for a high clinical potential and swift translation into the clinic. DisclosuresVrohlings: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Jungmichel: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Senn: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Howald: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Schleier: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Scheifele: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wendelspiess: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Richle: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Merten: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Lenherr-Frey: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Leisner: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA. Borras: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

P-006: Expanded natural killer cells with daratumumab, lenalidomide and dexamethasone combination potentiates antimyeloma activity in a myeloma xenograft model

<h3>Background</h3> Advances in the treatment of multiple myeloma (MM) significantly improved overall survival of patients. Nonetheless, MM still remains an incurable disease. This emphasizes urgent need for novel combination therapies for the treatment of MM. Recently, natural killer (NK) cells are becoming an attractive and safe tool in the field of immunotherapy. <h3>Methods</h3> Here, we investigated how daratumumab, lenalidomide, and dexamethasone (DRd) treatment potentiate antitumor effects of NK cells in a MM xenograft mouse model. NK cells were expanded and activated using K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells of MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNK), Rd, Rd + eNK, DRd, and DRd + eNK. DRd treatment strongly enhanced the cytotoxicity of eNK by upregulating NK cell's activation ligands and effector function. <h3>Results</h3> DRd combination with eNK significantly prolonged mouse survival and reduced MM progression and serum M-protein level. Moreover, DRd treatment significantly increased eNK persistence and homing into MM sites. <h3>Conclusions</h3> Our findings suggest that DRd treatment augments the anti-myeloma effects of ex vivo expanded and activated NK cells by modulating immune responses in MM-bearing mice.

Expanded natural killer cells augment the antimyeloma effect of daratumumab, bortezomib, and dexamethasone in a mouse model.

The use of natural killer (NK) cells is a promising and safe immunotherapeutic approach in the field of cancer immunotherapy. However, combination treatments are required to enhance the effector functions and therapeutic efficacy of NK cells. In this study, we investigated the potential of daratumumab (Dara), bortezomib, and dexamethasone (Dvd) to augment the antitumor effects of NK cells in a multiple myeloma (MM) xenograft mouse model. NK cells were expanded and activated using the K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells from MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor-bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNKs), Dara, Dara + eNKs, Dvd, and Dvd + eNKs. Dvd treatment strongly enhanced the cytotoxicity of eNKs by upregulating expression of NK cell activation ligands, downregulating expression of NK cell inhibitory ligands, and promoting antibody-dependent cellular cytotoxicity. The combination of eNKs with Dvd significantly prolonged mouse survival and reduced the tumor burden and serum M-protein level. Furthermore, Dvd pretreatment significantly increased eNK persistence and homing to MM sites. Our findings suggest that Dvd treatment potentiates the antimyeloma effects of NK cells expanded and activated ex vivo by modulating immune responses in MM-bearing mice.

The LncRNA RP11-301G19.1/miR-582-5p/HMGB2 axis modulates the proliferation and apoptosis of multiple myeloma cancer cells via the PI3K/AKT signalling pathway.

Long non-coding RNAs (lncRNAs) have recently been reported to act as crucial regulators and prognostic biomarkers of human tumorigenesis. Based on microarray data, RP11-301G19.1 was previously identified as an upregulated lncRNA during B cell development. However, the effect of RP11-301G19.1 on multiple myeloma (MM) cells remains unclear. In the present study, the effects of RP11-301G19.1 on tumour progression were ascertained both in vitro and in vivo. Our results demonstrated that RP11-301G19.1 was upregulated in MM cell lines and that its downregulation inhibited the proliferation and cell cycle progression and promoted the apoptosis of MM cells. Bioinformatic analysis and luciferase reporter assay results revealed that RP11-301G19.1 can upregulate the miR-582-5p-targeted gene HMGB2 as a competing endogenous RNA (ceRNA). Furthermore, Western blot results indicated that RP11-301G19.1 knockdown decreased the levels of PI3K and AKT phosphorylation without affecting their total protein levels. Additionally, in a xenograft model of human MM, RP11-301G19.1 knockdown significantly inhibited tumour growth by downregulating HMGB2. Overall, our data demonstrated that RP11-301G19.1 is involved in MM cell proliferation by sponging miR-582-5p and may serve as a therapeutic target for MM.

A Novel CD138-Targeting Monoclonal Antibody Induces Potent Myeloma Killing and Further Synergizes with IMiDs or Bortezomib in in Vitro and In Vivo Preclinical Models of Human Multiple Myeloma

Immunotherapeutically targeting CD138 has not been successfully translated into substantial clinical benefit in multiple myeloma (MM) patients. We here developed and determined in preclinical models of human MM the efficacy of VIS832, a novel humanized monoclonal antibody (MoAb) with differentiated CD138 target binding to the anti-CD138 MoAb BB4 within indatuximab ravtansine (BT082). Compared with BB4, VIS832 has significantly improved binding (P&amp;lt;.005, up to 1-log increases) to all MM cell lines (n&amp;gt;8). In the reporter-based antibody-dependent cellular cytotoxicity (ADCC) bioassay, EC50 of VIS832 was approximately 99.31 ng/ml (0.66 nM), whereas the BB4 possessing an isotype matched human IgG1 minimally induced ADCC against U266 MM cells. Evaluation of VIS832-induced ADCC in NK-MM cell co-cultures determined its EC50 values ranged from 2.22 + 0.37 to 15.3 + 2.71 ng/ml, with % maximal lysis of 37.06 + 1.45 % to 97.3 + 3.34 %, across tested MM cell lines (n=12), both sensitive or resistant to current therapies including dexamethasone, IMiDs, and bortezomib. While VIS832 effectively induced ADCC against parental cells with high and low CD138 levels, no lysis was detected in paired CD138-knockout MM cells. In the presence of BM stromal cells or osteoclasts, two key MM-supporting accessory cells in the BM milieu, VIS832-induced MM cytolysis was minimally affected. Importantly, VIS832 eliminated autologous MM cells of bone marrow (BM) samples from relapsed/refractory (RRMM) patients, with &amp;gt; 60% maximal lysis and &amp;gt; 90% killing in 4 out of 7 patient BM samples. EC50 values of VIS832 were 8.58-86.04 ng/ml in this patient cohort, comparable to those (17.28-179.3 ng/ml) of its non-humanized predecessor mAb 2810 against another RRMM patient cohort (n=6). In contrast to its potent autologous CD138+ patient cell lysis, VIS832 did not affect CD138-negative patient BM cells. VIS832 induced higher maximal lysis (~3-fold) of all target MM cell lines than CD38-targeting daratumumab (dara), regardless of resistance to lenalidomide and pomalidomide. These results also confirmed significantly higher CD138 vs CD38 levels in all MM cell lines and patient MM cells (P&amp;lt;.0001). Specifically, VIS832 remained active against MM cells resistant to dara, either via loss of CD38 or acquired drug resistance following extended dara treatment in exvivo co-cultures. Unlike dara, VIS832 selectively induced MM cell lysis via NK activation, without inducing NK cell apoptosis due to CD38 expression. When combined with lenalidomide, either pretreatment or added concomitantly, VIS832-induced MM cell lysis was significantly enhanced, with combination index (CI) of &amp;lt; 1 indicating synergism. Furthermore, antibody-dependent cellular phagocytosis (ADCP) represented another effector-mediated killing of VIS832 against MM cells, with phagocytosis dependent on both VIS832 and the E:T ratio. The ADCP activity of VIS832 was significantly higher than that observed for dara (P&amp;lt;.05) against MM cell lines sensitive or resistant to IMiDs and dara, and VIS832 induced significant ADCP against dara-resistant MM cells. Finally, in a disseminated MM1S xenograft model of human MM in CB-17 SCID mice, VIS832 alone reduced median tumor burden and improved overall survival from 30 to &amp;gt;60 days (P&amp;lt;.0001). This efficacy was sustained after discontinuation of treatment 3 weeks prior to study termination. Combined treatment of tumor-bearing mice with both VIS832 and bortezomib at sub-optimal doses rapidly eradicated tumors, resulting in 100% survival of all tumor-free animals to the end of study at day 73. The superior efficacy of combined VIS832 and bortezomib when compared to either agent alone suggests synergistic invivo activity, consistent with synergistically enhanced invitro cytotoxicity of this combination (CI &amp;lt; 1). Taken together, the significant invivo efficacy of VIS832, coupled with its mechanisms of action and potent in vitro MM cytotoxicity, strongly support clinical development of VIS832, as monotherapy and in combination, to overcome multidrug resistance and improve patient outcome in MM. Once its efficacy is established in RRMM, its favorable therapeutic index should allow for moving rapidly to earlier stages of disease, newly diagnosed MM and smoldering MM. Disclosures Chaganty: Visterra Inc: Current Employment. Ramakrishnan:Visterra Inc: Current Employment. Wollacott:Visterra Inc: Current Employment. Viswanathan:Visterra Inc: Current Employment. Adari:Visterra Inc: Current Employment. Munshi:Karyopharm: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BMS: Consultancy; Takeda: Consultancy; AbbVie: Consultancy. Babcock:Visterra Inc: Current Employment. Shriver:Visterra Inc: Current Employment. Myette:Visterra Inc: Current Employment. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Novel pyrrolobenzodiazepine benzofused hybrid molecules inhibit NF-κB activity and synergise with bortezomib and ibrutinib in hematological cancers.

Chronic lymphocytic leukemia (CLL) and multiple myeloma are incurable hematologic malignancies that are pathologically linked with aberrant nuclear factor-kappa B (NF-κB) activation. In this study, we identified a group of novel C8-linked benzofused pyrrolo[2,1- c][1,4]benzodiazepine monomeric hybrids capable of sequence-selective inhibition of NF-κB with low nanomolar LD50 values in CLL (n=46) and multiple myeloma cell lines (n=5). The lead compound, DC-1-192, significantly inhibited NF-κB DNA binding after just 4 h of exposure, demonstrating inhibitory effects on both canonical and non-canonical NF-κB subunits. In primary CLL cells, sensitivity to DC-1-192 was inversely correlated with RelA subunit expression (r2=0.2) and samples with BIRC3 or NOTCH1 mutations showed increased sensitivity (P=0.001). RNAsequencing and gene set enrichment analysis confirmed the over-representation of NF-κB regulated genes in the downregulated gene list. Furthermore, in vivo efficacy studies in NOD/SCID mice, using a systemic RPMI 8226 human multiple myeloma xenograft model, showed that DC- 1-192 significantly prolonged survival (P=0.017). In addition, DC1-192 showed synergy with bortezomib and ibrutinib; synergy with ibrutinib was enhanced when CLL cells were co-cultured on CD40L-expressing fibroblasts in order to mimic the cytoprotective lymph node microenvironment (P=0.01). Given that NF-κB plays a role in both bortezomib and ibrutinib resistance mechanisms, these data provide a strong rationale for the use of DC-1-192 in the treatment of NF-κB-driven cancers, particularly in the context of relapsed/refractory disease.

The JAK1/2 Inhibitor Ruxolitinib Downregulates CXCL12 Secretion from Bone Marrow Stromal Cells and M2 Macrophage Polarization in Multiple Myeloma

Introduction: The JAKSTAT pathway plays a critical role in the regulation of hematopoietic pathways and immunological cytokine signaling. The JAK pathway is also involved in tumor cell proliferation and drug resistance in multiple myeloma (MM). Thus, inhibition of the JAK pathway should be a potentially effective strategy for treating MM patients. B7-H3 is an immune checkpoint protein in the B7 superfamily and has been shown overexpressed in several tumors. Immune checkpoint blockade may suppress tumor progression or enhance anti-tumor immune responses. In this study, we investigated the effects of the JAK1/2 inhibitor ruxolitinib (Rux) on B7-H3 in MM. Materials and Methods: Bone marrow mononuclear cells (BMMCs) were collected from MM patients after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGλ-1A xenografts which had been grown in severe combined immunodeficient mice. HS-5 stromal and SUP-T1 T cells were purchased from ATCC. The cells were cultured and treated with or without RUX and then subjected to qRT-PCR, flow cytometric analysis, and western blot analysis. For qRT-PCR, total RNA was extracted and applied to cDNA synthesis, followed by qPCR. Gene expression was analyzed in MM BMMCs alone or co-cultured with stromal cells or T cells with or without Rux treatment (1μM) in vitro. Results: We identified increased B7-H3 expression in MMBMMCs from patients with progressive disease (PD) patients compared to those in complete remission (CR). Rux significantly reduced B7-H3 expression in MMBMMCs in patients with PD, MM cells (U266), and BM from patients in PD when co-cultured with stromal cells (HS-5) after 48-72 hours. Rux decreased B7-H3 expression in the human MM xenograft model LAGλ-1A when cultured ex vivo. In addition, Rux suppressed B7-H3 at protein levels as shown with flow cytometric analysis and western blotting, consistent with the gene expression results. Next, we tested whether B7-H3 blockade by Rux could potentially restore exhausted T cell activity against myeloma cells in MMBM. We found that Rux can increase IL-2 and CD8 gene expression in MMBM with lower plasma percentages (&lt; 30%) but not among those with higher plasma cell percentages (&gt;70%). Rux also elevated IL-2 and CD8 gene expression in BM when it was cocultured with T cells (SUP-T1), suggesting Rux may mediate immunological cytokine signaling. B7-H3-neutralizing antibody increased CD8 gene expression in MMBM in vitro, suggesting that one of the mechanisms through which Rux upregulates CD8 T cells in MMBM may be via downregulation of B7-H3. Conclusion: The immune checkpoint protein B7-H3 is overexpressed in MMBM in PD compared to CR patients. The JAK1/2 inhibitor Rux can decrease B7-H3 expression and increase IL-2 and CD8 expression in BM in vitro. Our results provide evidence for Rux inhibiting the immune checkpoint protein B7-H3 which may potentially restore exhausted T-cell activity in the MMBM tumoral microenvironment. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Takeda: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Sanofi: Consultancy.

The JAK1/2 Inhibitor Ruxolitinib Downregulates the Immune Checkpoint Protein B7-H3 in Multiple Myeloma

Introduction: The JAKSTAT pathway plays a critical role in the regulation of hematopoietic pathways and immunological cytokine signaling. The JAK pathway is also involved in tumor cell proliferation and drug resistance in multiple myeloma (MM). Thus, inhibition of the JAK pathway should be a potentially effective strategy for treating MM patients. B7-H3 is an immune checkpoint protein in the B7 superfamily and has been shown overexpressed in several tumors. Immune checkpoint blockade may suppress tumor progression or enhance anti-tumor immune responses. In this study, we investigated the effects of the JAK1/2 inhibitor ruxolitinib (Rux) on B7-H3 in MM. Materials and Methods: Bone marrow mononuclear cells (BMMCs) were collected from MM patients after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGλ-1A xenografts which had been grown in severe combined immunodeficient mice. HS-5 stromal and SUP-T1 T cells were purchased from ATCC. The cells were cultured and treated with or without RUX and then subjected to qRT-PCR, flow cytometric analysis, and western blot analysis. For qRT-PCR, total RNA was extracted and applied to cDNA synthesis, followed by qPCR. Gene expression was analyzed in MM BMMCs alone or co-cultured with stromal cells or T cells with or without Rux treatment (1μM) in vitro. Results: We identified increased B7-H3 expression in MMBMMCs from patients with progressive disease (PD) patients compared to those in complete remission (CR). Rux significantly reduced B7-H3 expression in MMBMMCs in patients with PD, MM cells (U266), and BM from patients in PD when co-cultured with stromal cells (HS-5) after 48-72 hours. Rux decreased B7H3 expression in the human MM xenograft model LAGλ-1A when cultured ex vivo. In addition, Rux suppressed B7-H3 at protein levels as shown with flow cytometric analysis and western blotting, consistent with the gene expression results. Next, we tested whether B7-H3 blockade by Rux could potentially restore exhausted T cell activity against myeloma cells in MMBM. We found that Rux can increase IL-2 and CD8 gene expression in MMBM with lower plasma percentages (&lt; 30%) but not among those with higher plasma cell percentages (&gt;70%). Rux also elevated IL-2 and CD8 gene expression in BM when it was cocultured with T cells (SUP-T1), suggesting Rux may mediate immunological cytokine signaling. B7-H3-neutralizing antibody increased CD8 gene expression in MMBM in vitro, suggesting that one of the mechanisms through which Rux upregulates CD8 T cells in MMBM may be via downregulation of B7-H3. Conclusion: The immune checkpoint protein B7-H3 is overexpressed in MMBM in PD compared to CR patients. The JAK1/2 inhibitor Rux can decrease B7-H3 expression and increase IL-2 and CD8 expression in BM in vitro. Our results provide evidence for Rux inhibiting the immune checkpoint protein B7-H3 which may potentially restore exhausted T-cell activity in the MMBM tumoral microenvironment. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:Amgen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Sanofi: Consultancy; Amag: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Takeda: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Replacement of miR-155 Elicits Tumor Suppressive Activity and Antagonizes Bortezomib Resistance in Multiple Myeloma.

Aberrant expression of microRNAs (miRNAs) has been associated to the pathogenesis of multiple myeloma (MM). While miR-155 is considered a therapeutic target in several malignancies, its role in MM is still unclear. The analysis of miR-155 expression indicates its down-regulation in MM patient-derived as compared to healthy plasma cells, thus pointing to a tumor suppressor role in this malignancy. On this finding, we investigated miR-155 replacement as a potential anti-tumor strategy in MM. The miR-155 enforced expression triggered anti-proliferative and pro-apoptotic effects in vitro. Given the lower miR-155 levels in bortezomib-resistant as compared to sensitive MM cells, we analyzed the possible involvement of miR-155 in bortezomib resistance. Importantly, miR-155 replacement enhanced bortezomib anti-tumor activity both in vitro and in vivo in a xenograft model of human MM. In primary MM cells, we observed an inverse correlation between miR-155 and the mRNA encoding the proteasome subunit gene PSMβ5, whose dysregulation has been largely implicated in bortezomib resistance, and we validated PSMβ5 3′UTR mRNA targeting, along with reduced proteasome activity, by miR-155. Collectively, our findings demonstrate that miR-155 elicits anti-MM activity, likely via proteasome inhibition, providing the framework for miR-155-based anti-MM therapeutic strategies.

Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription-PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

Patent highlights December 2016-January 2017.

Patent highlights December 2016-January 2017.

Lycorine Downregulates HMGB1 to Inhibit Autophagy and Enhances Bortezomib Activity in Multiple Myeloma

Multiple myeloma (MM) is the second most prevalent hematologic malignancy, characterized by the infiltration of malignant plasma cells into bone marrow. In spite of current efficient therapeutic regimens, which have significantly increased patients overall survival, the major features inevitably present in MM are the intrinsic and acquired resistance with nearly universal relapse. In addition, the diverse heterogeneous characteristics of this largely incurable disease emphasize the importance of innovative therapies and identification of more effective drugs. Autophagy removes defective cellular organelles, protein aggregates, and intracellular microbes and is associated with cell survival and tumor maintenance. Inhibition of autophagy enhances sensitivity of a number of anticancer agents and induces cell death in MM. High-mobility group box-1 (HMGB1) protein plays an important subcellular localization-dependent role during autophagy. The importance of HMGB1 for induction of autophagy and tumor development has made this protein as a novel target for cancer therapy. Lycorine is a natural alkaloid with significant anti-cancer activity. While previous studies mainly showed lycorine as a potential apoptosis inducer, recent studies stated that apoptosis is not the primary underlying anti-proliferative mechanism of this compound. This led the interest to investigate the role of lycorine on other cell maintenance systems, such as autophagy. In addition single-agent efficacy of lycorine or in combination with other anti-MM agents has not been evaluated in vivo. Herein we investigated the anti-MM effect of lycorine and the role of this natural agent on regulation of autophagy in vitro and in vivo. We found that lycorine inhibits proliferation and induces apoptosis in MM cells with less sensitivity to the normal B-cell at the same concentrations. We also found that lycorine promisingly inhibits autophagy, the mechanism that MM cells use to survive and defeat treatment. We identified HMGB1, an important regulator of autophagy, as the most aberrantly expressed protein after lycorine treatment. Furthermore, we characterized HMGB1 as a critical mediator of lycorine activity against MM. Gene expression profiling (GEP) analysis showed that higher expression of HMGB1 is linked with the poor prognosis of MM. We further confirmed this correlation in human bone marrow CD138+ primary myeloma cells and MM cell lines. Mechanistically, by activating the proteasomal degradation of HMGB1, lycorine induces a rapid turnover of HMGB1. This led to decreased Bcl-2 phosphorylation by MEK-ERK pathway and increased association of Bcl-2 with Beclin-1 resulting in autophagy inhibition and growth attenuation. In addition, we observed higher HMGB1 expression in bortezomib resistant cells. The combination of bortezomib plus lycorine was highly efficient against MM cells and MM cells grown in bone marrow micro-environment. Lycorine showed the capability of inhibiting bortezomib induced autophagy as well as re-sensitizing resistant cells to bortezomib. In agreement with our in vitro observations, in vivo study using human MM xenograft model showed that lycorine is well tolerated, inhibits HMGB1 expression and thereby autophagy and induces enhanced bortezomib activity. These observations indicated lycorine as an effective autophagy inhibitor and revealed that lycorine alone or in combination with bortezomib is a potential therapeutic strategy. Our study supports the feasibility of lycorine in anti-MM treatment and provides a preclinical framework for combining lycorine with bortezomib in clinical setting. DisclosuresNo relevant conflicts of interest to declare.

A Novel Agent SL-401 Triggers Anti-Myeloma Activity By Targeting Plasmacytoid Dendritic Cells: Implications for a Novel Immune-Associated Mechanism

Introduction Multiple myeloma (MM) remains incurable despite novel therapies, highlighting the need for further identification of factors mediating disease progression and resistance. Our studies have identified an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. The functional significance of increased numbers of pDCs in MM BM is evident from our observations that pDCs: are relatively resistant to novel and conventional therapies; protect tumor cells from therapy-induced cytotoxicity; promote tumor growth and survival; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323). Aberrant pDC function is evidenced in their interactions not only with MM cells, but also with other immune effector T cells and NK cells in the MM BM milieu (Ray et al, Leukemia 2015, 29:1441-1444). Directly targeting pDC interactions with MM and immune effector cells in the MM BM milieu will be required to enhance both anti-tumor immunity and cytotoxicity. However, therapies targeting pDCs are lacking. We found that IL-3R is highly expressed on pDCs, and that pDC-MM interactions trigger secretion of IL-3, which in turn, promotes both pDC survival and osteolytic bone disease. Recent efforts have led to the development of a novel therapeutic agent SL-401, which specifically targets IL-3R-expressing pDCs. Here we examined the effect of SL-401 on pDC-induced MM cell growth both in vitro and in vivo, as well as on IL-3R-expressing osteoclasts.Methods Patient MM cells, pDCs, and MNCs were obtained from normal donors or MM patients. Cell growth/viability was analyzed using MTT/WST assays. OCL function and bone resorption were measured using the OsteoAssays and TRAP staining. The RPMI-8226 cell line was used to isolate MM-SPs by flow-cytometry based Hoechst 33342 staining. SL-401 is a recombinant protein expressed in E. coli. The hybrid gene is comprised of human IL-3 fused to truncated diphtheria toxin (DT). The IL-3 domain of SL-401, which replaces the native binding domain of DT, targets SL-401 to cells that overexpress IL-3R. SL-401 was obtained from Stemline Therapeutics, NY; bortezomib, lenalidomide, pomalidomide, and melphalan were purchased from Selleck Chemicals. For animal model studies, SL-401 (16.5 μg/kg) was administered intravenously daily for 2 weeks.Results SL-401 triggered significant apoptosis in pDCs (>95%) at low picomolar concentrations that are well within clinically achievable doses.Higher concentrations of SL-401 trigger a modest apoptosis (30%± 1.3% apoptosis at 83 ng/ml or 1.3 nM) in MM cells due to lower IL-3R expression versus pDCs. Moreover, SL-401 did not significantly induce apoptosis of normal PBMCs (8% ± 0.5% apoptosis at 83 ng/ml), suggesting a favorable therapeutic index for SL-401. SL-401 inhibited pDC-induced growth of MM cell lines and patient MM cells in a dose-dependent manner. Moreover, 6 of 9 MM samples were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Combinations of SL-401 with melphalan, bortezomib, lenalidomide, or pomalidomide induced synergistic anti-MM activity (Combination index < 1). SL-401 blocked monocyte-derived osteoclast formation in a dose-dependent fashion, as well as restored MM patient BM-derived osteoblast formation. Having defined the efficacy of SL-401 in targeting pDCs and pDC-triggered MM cell growth in vitro, we validated these findings in vivo using our murine xenograft model of human MM, under auspices of protocols approved by our institutional animal protection committee. SL-401 inhibited pDC-induced MM cell growth in vivo and prolonged survival in a murine xenograft model of human MM. We also evaluated the efficacy of SL-401 in vivo using our SCID-human (SCID-hu) mouse model, which reflects the human BM milieu with human cytokines and extracellular matrix proteins. SL-401 significantly abrogated pDC-triggered MM cell growth in vivo in SCID-hu model.Conclusions Our data provide the basis for using SL-401 to directly target pDCs and inhibit the pDC-MM interaction as well as target osteolytic bone disease in novel therapeutic strategies in order to enhance MM cytotoxicity, overcome drug resistance, and improve patient outcome. The interactions of immune effector cells in the MM tumor microenvironment also provide a rationale for combining SL-401 with checkpoint inhibitors.Correspondence: Dharminder Chauhan DisclosuresMacri:Stemline Therapeutics, Inc., New York, NY USA: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Richardson:Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Chauhan:Stemline Therapeutics: Consultancy.