Anti-multiple Myeloma Effects Research Articles

Hexamethylene amiloride induces lysosome-mediated cell death in multiple myeloma through transcription factor E3

Multiple myeloma (MM) is the second common hematological malignancy characterized by the abnormal proliferation of plasma cells. Although advances in the past decades have led to improved outcomes and longer survival, MM remains largely incurable. New targets and targeted therapy may help to achieve better outcomes. Proton exporter NHE1 is highly expressed by tumor cells to maintain pH gradient for their survival and its inhibitor Hexamethylene amiloride (HA) has been demonstrated anti-tumor effect. However, whether HA could inhibit MM remains unknown. In this study, we firstly demonstrated that elevated expression level of NHE1 is associated with poor prognosis of MM. Moreover, the NHE1 inhibitor HA inhibited growth and induced apoptosis effectively in both MM cell lines and primary bone marrow cells from MM patients. Mechanistically, inhibitory effect was achieved partially through TFE3-mediated lysosomal production. With a MM xenograft mouse model, we verified that HA has a significant anti MM effect in vivo. Importantly, HA induced apoptosis of the carfilzomib-resistant MM cells and enhanced the effect of carfilzomib in MM. In summary, we demonstrated that NHE1 inhibitor HA can effectively inhibit MM growth both in vitro and in vivo, providing a new therapeutic strategy for improved outcome of de novo and resistant MM.

A Phase I Trial Evaluating the Addition of Lenalidomide to Patients with Relapsed/Refractory Multiple Myeloma Progressing on Ruxolitinib and Methylprednisolone.

Ruxolitinib (RUX), an orally administered selective Janus kinase 1/2 inhibitor, has received approval for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. We have previously demonstrated the anti-multiple myeloma effects of RUX alone and in combination with the immunomodulatory agent lenalidomide (LEN) and glucocorticosteroids both pre-clinically and clinically. This study aims to evaluate whether LEN can achieve clinical activity among patients with multiple myeloma progressing on the combination of RUX and methylprednisolone (MP). In this part of a phase I, multicenter, open-label study, we evaluated the safety and efficacy of RUX and MP for patients with multiple myeloma with progressive disease who had previously received a proteasome inhibitor, LEN, glucocorticosteroids, and at least three prior regimens; we also determined the safety and efficacy of adding LEN at the time of disease progression from the initial doublet treatment. Initially, all subjects received oral RUX 15 mg twice daily and oral MP 40 mg every other day. Those patients who developed progressive disease according to the International Myeloma Working Group criteria then received LEN 10 mg once daily on days 1-21 within a 28-day cycle in addition to RUX and MP, which were administered at the same doses these patients were receiving at the time progressive disease developed. Twenty-nine subjects (median age 64 years; 18 [62%] male) were enrolled in this part of the study and initially received the two-drug combination of RUX and MP. The median number of prior therapies was six (range 3-12). The overall response rate from this two-drug combination was 31% and the clinical benefit rate was 34%. The best responses were 1 very good partial response, 8 partial responses, 1 minor response, 12 stable disease, and 7 progressive disease. The median progression-free survival was 3.5 months (range 0.5-36.2months). The median time to response was 3.0 months. The median duration of response was 12.5 months (range 2.8-36.2 months). Twenty (69%) patients whoshowed progressive disease had LEN added to RUX and MP; all patients had prior exposure to LEN and all but one patient was refractory to their last LEN-containing regimen. After the addition of LEN, the overall response rate was 30% and the clinical benefit rate was 40%. The best responses of patients following the addition of LEN were 2 very good partial responses, 4 partial responses, 2 minor responses, 8 stable disease, and 4 progressive disease. The median time to response was 2.6 months (range 0.7-15.0 months). The median duration of response was not reached. The median progression-free survival following the addition of LEN was 3.5 months (range 0.3-25.9 months). For patients with multiple myeloma, treatment with RUX and MP is effective and well tolerated, and LEN can be used to extend the benefit of this RUX-based treatment. This study is registered with ClinicalTrials.gov, NCT03110822, and is ongoing.

A Phase 1 Trial Evaluating the Addition of Lenalidomide to Relapsed/Refractory Multiple Myeloma Patients Progressing on Ruxolitinib and Methylprednisolone

Background Ruxolitinib (RUX), an orally administered selective JAK 1/2 inhibitor, has received approval for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. We have previously demonstrated the anti-multiple myeloma (MM) effects of RUX alone and in combination with the immunomodulatory agent lenalidomide (LEN) and glucocorticosteroids both preclinically and clinically. Although we have previously demonstrated that LEN and steroid refractory MM patients respond to the addition of RUX, no studies have evaluated whether LEN can achieve clinical activity among those progressing on the combination of RUX and methylprednisolone. Methods Patients included those with MM showing progressive disease (PD) who had previously received a proteasome inhibitor, LEN, glucocorticosteroids, and at least three prior regimens. Initially, subjects received oral RUX 15 mg twice daily and oral methylprednisolone 40 mg every other day. Those patients who developed PD according to International Myeloma Working Group criteria had LEN 10 mg once daily days 1-21 on a 28-day cycle added to RUX and methylprednisolone which were administered at the same doses they were receiving at the time of PD. Results A total of 29 subjects were enrolled and initially received the combination of RUX and methylprednisolone. Follow up was until June 30, 2023. The median number of prior therapies was 6 (range, 3-12). The overall response rate (ORR) was 31% and the clinical benefit rate (CBR) was 34%. The best responses were 1 very good partial response (VGPR), 8 partial responses (PR), 1 minor response (MR), 12 stable disease (SD), and 7 PD. The median progression-free survival (PFS) was 3.5 mo (range, 0.5-36.2 mo). The median time to response was 3.0 mo (n=10). The median duration of response (DOR) was 12.5 mo (range, 2.8-36.2 mo). Twenty (69%) showed PD and had LEN added to the RUX and methylprednisolone combination: all of these patients were refractory to their last LEN-containing regimen. After the addition of LEN, the ORR was 30% and CBR was 40%. The best responses of patients following the addition of LEN were 2 VGPR, 4 PR, 2 MR, 8 SD, and 4 PD. The majority of patients (71%) who initially achieved a > MR to the two-drug combination responded following the addition of LEN whereas only 23% of patients who did not initially achieve a > MR responded to the addition of the third drug. The median time to response was 2.6 mo (range, 0.7-15 mo). The median DOR was undefined (range, 1.2-10.9 mo). The median PFS was 3.5 mo (range, 0.3-25.9 mo). The median PFS2 (including time from first treatment to end of study or June 30, 2023) was 7.3 mo (range, 0.5-39.9). Treatment was well tolerated with no additional toxicity identified following the addition of LEN. Interestingly, PFS2 was predicted by the patient's baseline serum B-cell maturation level; patients with values above the median (128 ng/mL) had significantly shorter PFS2 than those below it (3.5 mo vs. undefined, p = 0.0337). Conclusion The addition of LEN to heavily, previously treated relapsed/refractory MM patients progressing on RUX and methylprednisolone shows significant clinical activity. This three-drug combination is well tolerated and is able to extend the benefit of RUX-based treatment for these patients. The promising results from this phase 1 clinical trial demonstrate a potential novel, all oral therapeutic approach for treating RRMM patients.

Targeting Bclxl Mitigates Mcl1 Chemoresistance in Multiple Myeloma

Aim:Despite the adoption of novel therapeutic modalities, Multiple Myeloma (MM) remains incurable. The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. These results underscore the significance of Bcl2 alternative anti-apoptotic proteins (Mcl1 and BclxL) for the survival of myeloma cells.Method:We validated the anti MM effect of the Mcl1 inhibitor S63845 both in vitro utilising 11 human myeloma cell lines (HMCL) and ex vivo against n=30 primary MM tumours. Comparative analysis of RNAseq between S63845 sensitive and resistant HMCL was undertaken to identify candidate proteins that potentially modulate resistance to S63845. Treatment with S63845 and rationally selected combination partners was further evaluated in vitro, ex vivo and in vivo with flow cytometry, immunoblotting and live imaging mitochondria fitness monitoring.Results:RNAseq identified BclxL as potential mediator of resistance to S63845 in HMCL. Immunoblotting confirmed high BclxL expression and high BclxL/BclS in S63845 resistant HMCL. Five S63845 resistant HMCL (U266, ANBL6, KMS28PE, EJM, MM1R) and primary tumours were treated with S63845 combined with the BclxL inhibitor A1331852 . Combined treatment of the HMCL demonstrated a high Bliss synergy score for all the HMCL tested (54, 42, 24, 47, 45 for U266, EJM, KMS28PE, MM1R and ANBL6 respectively) and induced synergistic killing of 80% of the primary tumours treated. Dual inhibition in U266 induced an 80% drop in intracellular ATP at 4h with an increase in active Caspases 9 and 8 (4.5 and 5 fold, respectively). Similarly, the combination induced a 78% drop in mitochondrial transmembrane potential (TMRE intensity) by 4h with live imaging revealing striking mitochondrial damage as early as 40 minutes after exposure (figure). These changes were associated with a reduction of both Mcl1 and, BclxL proteins and Bim and Bid protein levels. No changes were seen in the level of Bcl2, Bak or Bax protein expression. The combination of S63845 and A1331852 in healthy NSG mice at 12.5mg/kg proved lethal due to hepatotoxicity, arguing against the clinical utility of such an approach. However, this observed anti-MM synergistic activity was recapitulated when S63845 was combined with the already approved anti-MM therapeutic panobinostat, with the induction of a significant reduction in both BclxL and Myc protein levels at 24h, and synergistic killing of 56% of primary tumours.Conclusion:High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo. Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation. [Display omitted] DisclosuresSpencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Isatuximab in Combination with Chemotherapy in Pediatric Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia or Acute Myeloid Leukemia (ISAKIDS): Interim Analysis

Isatuximab in Combination with Chemotherapy in Pediatric Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia or Acute Myeloid Leukemia (ISAKIDS): Interim Analysis

Blocking HDAC3 in Bone Marrow Stromal Cells Has Direct Anti-Multiple Myeloma Effect and Modulates T Cell Function

IntroductionHistone deacetylases (HDAC) are therapeutic targets in Multiple Myeloma (MM) and the pan-HDAC inhibitor Panobinostat is FDA approved. However, clinical trials showed that non-selective HDAC inhibitors have a narrow therapeutic index, prompting the development of isoform-specific HDAC inhibitors such as the HDAC6 inhibitor Ricolinostat. Preclinical studies have shown that HDAC3 downregulation/inhibition exerts an anti-myeloma effect, suggesting that HDAC3 may be a novel molecular target in MM. However, the effect of targeting HDAC3 in the bone marrow (BM) microenvironment has not been investigated. The BM microenvironment, particularly stromal and immune cells, have been shown to be key in supporting MM growth, immune escape, and resistance to therapy. We sought to study whether targeting HDAC3 in BM stroma cells (BMSC) has direct cytotoxic effects against stromal cells and/or impacts MM cell and T cell function through secreted factors and/or cell-to-cell contact mechanisms.MethodsWe targeted HDAC3 in human BMSC line HS-5 and primary-derived BMSC from MM patients (MM-BMSC) via siRNA or via the HDAC3-selective inhibitor BG45. MTT assay and flow cytometry with annexin V/PI staining were used to assess viability and apoptosis post-HDAC3 KD/pharmacologic inhibition. Downregulation of HDAC3 was confirmed by immunoblotting. To assess the effect of HDAC3 silencing in MM-BMSC on MM cells, we performed siRNA-mediated HDAC3 KD in HS-5 and MM-BMSC, removed transfection media after 48 hours, and performed co-culture experiments with MM1S.Luc and H929.Luc MM cell lines. After 96 hours MM cell proliferation was measured using the Luciferase Assay. Scrambled siRNA transfected HS-5 and MM-BMSC were used as control. To assess the effect of HDAC3 silencing in BMSC on T cells, we isolated peripheral blood mononuclear cells from healthy donors by Ficoll density gradient separation. CD3+ T cells were isolated using EasySep™ Human T Cell Isolation Kit and activated using Dynabeads Human T-Activator CD3/CD28. Cytokine array profiling was performed on the cell-culture supernatant using the Raybiotech C-series Human Cytokine Array according to manufacturer's protocol.ResultsFirst, we showed that pharmacological inhibition of HDAC3 via BG45 does not have direct anti-proliferative or cytotoxic effect on the HS-5 stromal cell line at doses up to 5-fold higher than the EC50 of MM cell lines. Consistent with this data, siRNA-mediated HDAC3 KD does not have direct anti-proliferative and/or cytotoxic effects on HS-5 or MM-BMSC. However, when MM cell lines were co-cultured with HDAC3-silenced HS-5 or MM-BMSC, there was a statistically significant decrease in BMSC-induced MM proliferation compared to control (HS-5: 38% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.1-27.5% decrease in MM proliferation in HDAC3 KD, p < 0.05). To assess whether cell-to-cell contact is necessary to mediate this growth arrest, we cultured MM cell lines in conditioned media from HDAC3 or scrambled siRNA transfected BMSC and discovered that the former was sufficient to induce growth/proliferation arrest (HS-5: 31.5% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.3% decrease in MM proliferation in HDAC3 KD, p < 0.05), suggesting a paracrine effect.We next showed that conditioned supernatant from HDAC3-KD HS-5, cultured alone or in combination with MM cell lines, significantly inhibited the proliferation of activated T-cells (HS-5 supernatant: 47.8% decrease in MM proliferation in HDAC3 KD; HS-5 + MM co-culture supernatant: 32.6% decrease in MM proliferation in HDAC3 KD). To identify the soluble factors responsible for this paracrine effect, we subjected HDAC3-KD and scrambled-transfected HS-5 supernatant to cytokine profiling. We identified a number of cytokines modulated by HDAC3 KD, which we are currently analyzing. Among these, we identified a 1.37-fold increase in soluble PD-L1 when HDAC3 was silenced in HS-5 cells.ConclusionsOur results show that inhibiting HDAC3 in BMSC mediates anti-MM and anti-T cell effect via soluble factors. Gene expression profiling and proteomic studies are currently underway to define the molecular mechanisms supporting these effects. Our preliminary data suggest that targeting HDAC3 in BMSC may alter the checkpoint repertoire in the BM milieu, suggesting potential utility of therapeutic strategies combining HDAC3 and checkpoint inhibitors. DisclosuresHideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Mazitschek:Acetylon: Equity Ownership. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

Allogeneic Stem Cell Transplantation in Patients with High-Risk Multiple Myeloma: Utopia or Continuous Challenge in Aiming for Cure?

Nowadays, several novel agents have been introduced in the treatment of multiple myeloma, not only resulting in high response rates and prolonged survival but also offering good quality of life. However, the potential of cure, especially for patients with advanced or unfavorable disease features, remains elusive. Allogeneic hematopoietic stem cell transplantation, based mainly on the graft vs. myeloma effect, can offer prolonged disease control and probability of cure but unfortunately at the cost of considerable transplant-related toxicity rates. Therefore, the role of allogeneic hematopoietic stem cell transplantation in the treatment of multiple myeloma has been called into question. Recently, several studies, particularly those with long-term follow-up, demonstrated a trend of survival superiority for allografted patients with high-risk disease. These data fuel again the interest in allogeneic stem cell transplantation for selected patients with high-risk multiple myeloma, especially if the high remission rates which can be achieved with the currently used treatment protocols could be long-life sustained through the additional exploitation of the long-lasting anti-multiple myeloma effect, originating from the allograft.

Directly targeting c-Myc contributes to the anti-multiple myeloma effect of anlotinib

Despite the significant advances in the treatment of multiple myeloma (MM), this disease is still considered incurable because of relapse and chemotherapy resistance, underscoring the need to seek novel therapies with different mechanisms. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor (TKI), has exhibited encouraging antitumor activity in several preclinical and clinical trials, but its effect on MM has not been studied yet. In this study, we found that anlotinib exhibits encouraging cytotoxicity in MM cells, overcomes the protective effect of the bone marrow microenvironment and suppresses tumor growth in the MM mouse xenograft model. We further examined the underlying molecular mechanism and found that anlotinib provokes cell cycle arrest, induces apoptosis and inhibits multiple signaling pathways. Importantly, we identify c-Myc as a novel direct target of anlotinib. The enhanced ubiquitin proteasomal degradation of c-Myc contributes to the cell apoptosis induced by anlotinib. In addition, anlotinib also displays strong cytotoxicity against bortezomib-resistant MM cells. Our study demonstrates the extraordinary anti-MM effect of anlotinib both in vitro and in vivo, which provides solid evidence and a promising rationale for future clinical application of anlotinib in the treatment of human MM.

Anti-Cancer Activity of Curcumin on Multiple Myeloma

Multiple Myeloma (MM) is the third most common and deadly hematological malignancy, which is characterized by a progressive monoclonal proliferation within the bone marrow. MM is cytogenetically heterogeneous with numerous genetic and epigenetic alterations, which lead to a wide spectrum of signaling pathways and cell cycle checkpoint aberrations. MM symptoms can be attributed to CRAB features (hyperCalcemia, Renal failure, Anemia, and Bone lesion), which profoundly affect both the Health-Related Quality of Life (HRQoL) and the life expectancy of patients. Despite all enhancement and improvement in therapeutic strategies, MM is almost incurable, and patients suffering from this disease eventually relapse. Curcumin is an active and non-toxic phenolic compound, isolated from the rhizome of Curcuma longa L. It has been widely studied and has a confirmed broad range of therapeutic properties, especially anti-cancer activity, and others, including anti-proliferation, anti-angiogenesis, antioxidant and anti-mutation activities. Curcumin induces apoptosis in cancerous cells and prevents Multidrug Resistance (MDR). Growing evidence concerning the therapeutic properties of curcumin caused a pharmacological impact on MM. It is confirmed that curcumin interferes with various signaling pathways and cell cycle checkpoints, and with oncogenes. In this paper, we summarized the anti- MM effects of curcumin.

Atractylenolide I Can Induce Apoptosis of U266 Cells and Enhance Bortezomib Effect

To investigate the effect of atractylenolide I on proliferation and apoptosis of U266 cells, and anti-multiple myeloma effect of bortezomib. Bortezomib, bortezomib combined atractylenolide I and atractylenolide I at different concentrations were added into U266 cells respectively, cellular proliferation toxicity was evaluated by CCK-8 assay, apoptosis and cell cycle were detected by using flow cytometry with Annexin V-FITC/PI staining. RT-PCR and Western blot analysis were used to detect the mRNA and protein levels of targeting gene Caspase-3,Caspase-9,BCL-2,BAX,JAK2,STAT3 and IL-6, respectively. The proliferation of U266 cells could inhibited by atractylenolide I, and the apoptosis of U266 cells could be promoted by atractylenolide I, also, which showed a dose-dependent manner(P<0.00; r=0.99). Moreover, the atractylenolide I could regulat the mitochondrial pathway(P<0.01). The combination of 2 drugs could strengther the inhibition of U266 cell proliferation significantly, and the expression level of IL-6,JAK2,STAT3 and BCL-2 mRNA and protein could be decreased by single drug and 2 drugs both(P<0.01). Atractylenolide I significantly inhibits the proliferation of U266 cells and promotes their apoptosis. At the same time, it acts synergistically with bortezomib, which may be related to mitochondrial pathway, and probably related to the regulating of IL-6, JAK2 and STAT3 gene expression in signal pathway of JAK2/STAT3.

Atractylenolide I Can Induce Apoptosis of U266 Cells and Enhance Bortezomib Effect

To investigate the effect of atractylenolide I on proliferation and apoptosis of U266 cells, and anti-multiple myeloma effect of bortezomib. Bortezomib, bortezomib combined atractylenolide I and atractylenolide I at different concentrations were added into U266 cells respectively, cellular proliferation toxicity was evaluated by CCK-8 assay, apoptosis and cell cycle were detected by using flow cytometry with Annexin V-FITC/PI staining. RT-PCR and Western blot analysis were used to detect the mRNA and protein levels of targeting gene Caspase-3,Caspase-9,BCL-2,BAX,JAK2,STAT3 and IL-6, respectively. The proliferation of U266 cells could inhibited by atractylenolide I, and the apoptosis of U266 cells could be promoted by atractylenolide I, also, which showed a dose-dependent manner(P<0.00; r=0.99). Moreover, the atractylenolide I could regulat the mitochondrial pathway(P<0.01). The combination of 2 drugs could strengther the inhibition of U266 cell proliferation significantly, and the expression level of IL-6,JAK2,STAT3 and BCL-2 mRNA and protein could be decreased by single drug and 2 drugs both(P<0.01). Atractylenolide I significantly inhibits the proliferation of U266 cells and promotes their apoptosis. At the same time, it acts synergistically with bortezomib, which may be related to mitochondrial pathway, and probably related to the regulating of IL-6, JAK2 and STAT3 gene expression in signal pathway of JAK2/STAT3.

Maternal embryonic leucine zipper kinase inhibitor OTSSP167 has preclinical activity in multiple myeloma bone disease.

Multiple myeloma bone disease is characterized by an uncoupling of bone remodeling in the multiple myeloma microenvironment, resulting in the development of lytic bone lesions. Most myeloma patients suffer from these bone lesions, which not only cause morbidity but also negatively impact survival. The development of novel therapies, ideally with a combined anti-resorptive and bone-anabolic effect, is of great interest because lesions persist with the current standard of care, even in patients in complete remission. We have previously shown that MELK plays a central role in proliferation-associated high-risk multiple myeloma and its inhibition with OTSSP167 resulted in decreased tumor load. MELK inhibition in bone cells has not yet been explored, although some reports suggest that factors downstream of MELK stimulate osteoclast activity and inhibit osteoblast activity, which makes MELK inhibition a promising therapeutic approach. Therefore, we assessed the effect of OTSSP167 on bone cell activity and the development of myeloma-induced bone disease. OTSSP167 inhibited osteoclast activity in vitro by decreasing progenitor viability as well as via a direct anti-resorptive effect on mature osteoclasts. In addition, OTSSP167 stimulated matrix deposition and mineralization by osteoblasts in vitro. This combined anti-resorptive and osteoblast-stimulating effect of OTSSP167 resulted in the complete prevention of lytic lesions and bone loss in myeloma-bearing mice. Immunohistomorphometric analyses corroborated our in vitro findings. In conclusion, we show that OTSSP167 has a direct effect on myeloma-induced bone disease in addition to its anti-multiple myeloma effect, which warrants further clinical development of MELK inhibition in multiple myeloma.

Adverse Metaphase Cytogenetics Can Be Overcome by Adding Bortezomib and Thalidomide to Fractionated Melphalan Transplants.

Purpose: To determine whether a reduction in the intensity of Total Therapy (TT) reduces toxicity and maintains efficacy.Experimental Design: A total of 289 patients with gene expression profiling (GEP70)-defined low-risk multiple myeloma were randomized between a standard arm (TT4-S) and a light arm (TT4-L). TT4-L employed one instead of two inductions and consolidations. To compensate for potential loss of efficacy of TT4-L, bortezomib and thalidomide were added to fractionated melphalan 50 mg/m2/d for 4 days.Results: Grade ≥3 toxicities and treatment-related mortalities were not reduced in TT4-L. Complete response (CR) rates were virtually identical (P = 0.2; TT4-S, 59%; TT4-L, 61% at 2 years), although CR duration was superior with TT4-S (P = 0.05; TT4-S, 87%; TT4-L, 81% at 2 years). With a median follow-up of 4.5 years, there was no difference in overall survival (OS) and progression-free survival (PFS). Whereas metaphase cytogenetic abnormalities (CAs) tended to be an adverse feature in TT4-S, as with predecessor TT trials, the reverse applied to TT4-L. Employing historical TT3a as training and TT3b as test set, 51 gene probes (GEP51) significantly differentiated the presence and absence of CA (q < 0.0001), seven of which function in DNA replication, recombination, and repair. Applying the GEP51 model to clinical outcomes, OS and PFS were significantly inferior with GEP51/CA in TT4-S; such a difference was not observed in TT4-L.Conclusions: We identified a prognostic CA-linked GEP51 signature, the adversity of which could be overcome by potentially synergizing anti-multiple myeloma effects of melphalan and bortezomib. These exploratory findings require confirmation in a prospective randomized trial. Clin Cancer Res; 23(11); 2665-72. ©2016 AACR.

Combined TRAF6 Targeting and Proteasome Blockade Has Anti-myeloma and Anti-Bone Resorptive Effects

TNF receptor-associated factor 6 (TRAF6) has been implicated in polyubiquitin-mediated IL1R/TLR signaling through activation of IκB kinase (IKK) to regulate the NF-κB and JNK signaling pathways. Here, TRAF6 protein was determined to be overexpressed in bone marrow mononuclear cells (BMMC) from patients with multiple myeloma. TRAF6 expression in BMMCs from patients with progressive disease is significantly elevated as compared with individuals in complete remission, with monoclonal gammopathy of undetermined significance, or healthy subjects. Furthermore, TRAF6 dominant-negative (TRAF6dn) peptides were constructed which specifically reduced TRAF6 signaling and activation of IKK. TRAF6 not only reduced cellular growth but also increased the apoptosis of multiple myeloma tumor cells in a concentration-dependent fashion. Because TRAF6 activates IKK through polyubiquitination, independent of its proteasome activity, a TRAF6dn peptide was combined with the proteasome inhibitors bortezomib or carfilzomib to treat multiple myeloma. Importantly, targeting of TRAF6 in the presence of proteasome inhibition enhanced anti-multiple myeloma effects and also decreased TLR/TRAF6/NF-κB-related signaling. Finally, TRAF6dn dose dependently inhibited osteoclast cell formation from CD14+ monocytes, induced with RANKL and mCSF, and markedly reduced bone resorption in dentin pits. In all, these data demonstrate that blocking TRAF6 signaling has anti-multiple myeloma effects and reduces bone loss.Implications: The ability to target TRAF6 signaling and associated pathways in multiple myeloma suggests a promising new therapeutic approach. Mol Cancer Res; 15(5); 598-609. ©2017 AACR.

Repurposing Leflunomide for Multiple Myeloma Treatment

Background: Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide, a commercially available oral immunosuppressive agent that has been FDA-approved since 1998 for the treatment of rheumatoid arthritis (RA) was evaluated as a potential MM therapy. The primary mechanism of action is de novo inhibition of pyrimidine synthesis by targeting dihydroorotate dehydrogenase (DHODH), and thus achieving an anti-proliferative effect in B- and T-lymphocytes. A secondary mechanism of action is inhibition of cytokine and growth factor receptor-associated tyrosine kinase activity. Methods and Results: Pre-clinical studies of teriflunomide, the active metabolite of leflunomide, showed that it inhibited cell growth and induced apoptosis in MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226) and primary MM patients’ (CD138+) plasma cells at clinically achievable concentrations (50-200 uM) in a time- and dose-dependent manner. We also found that teriflunomide induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <200 uM. In addition, teriflunomide and dexamethasone synergized in the in vitro growth inhibition of MM cell line MM.1S. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon teriflunomide exposure, MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary plasma cells from two MM patient samples were treated with 200 µM teriflunomide or DMSO control for 24 h before extraction and purification of mRNA and microRNA. mRNA-seq and miRNA-seq analysis from teriflunomide-treated MM samples revealed that similar changes were present between patient samples and cell lines. A total of 382 genes were found to be differentially expressed (225 upregulated, 157 downregulated). Upregulated genes included those that participate in defense response and negative regulation of cell growth. Genes involved in mitosis, rRNA biogenesis/processing, and immune response were generally downregulated. Analysis of microRNA-seq data from these samples revealed five differentially expressed, mostly newly discovered miRNAs that have unknown function.Conclusions: Leflunomide and its analogues demonstrated anti MM effects in vitro as well as synergy with dexamethasone. Based on our promising pre-clinical results we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory MM. DisclosuresNo relevant conflicts of interest to declare.

A GRP78-Directed Monoclonal Antibody Recaptures Response in Refractory Multiple Myeloma with Extramedullary Involvement.

Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma, and both its surface expression and its biologic significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface GRP78 and leads to disease stabilization when used as single agent in a clinical trial. In this article, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens. GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of multiple myeloma. Activity of PAT-SM6 was evaluated in combination with anti-multiple myeloma agents lenalidomide, bortezomib, and dexamethasone in vitro Finally, we report on a multiple myeloma patient with relapsed-refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide. Although sGRP78 expression was present at all stages, it increased with disease progression and was even strongly elevated in patients with drug-resistant and extramedullary disease. Pretreatment with dexamethasone as well as dual combination of PAT-SM6/lenalidomide further increased sGRP78 expression and consecutively showed synergistic anti-multiple myeloma effects with PAT-SM6 in proliferation assays. As proof of concept, a 62-year-old male with triple resistant multiple myeloma treated with PAT-SM6, bortezomib, and lenalidomide experienced partial remission of both intra- and extramedullary lesions. PAT-SM6 therapy in combination regimens showed efficacy in relapsed-refractory multiple myeloma. Clin Cancer Res; 22(17); 4341-9. ©2016 AACR.

Curative Efficacy of Lenalidomide plus Low Dose Dexamethasone for Multiple Myeloma

To explore the clinical efficacy and safety of lenalidomide plus low dose dexamethasone for treating patients with multiple myeloma (MM). A total of 19 MM patients were enrolled to receive the therapeutic schedule of lenalidomide plus dexamethasone in our hospital from May 2013 to June 2015. Lenalidomide 25 mg was taken orally daily for 21 days and resting for 7 days, and dexamethasone 10 mg was taken orally daily on the day 1-4, 7-10 and 13-16. The regimens were Rd (lenalidomide and dexamethasone, n = 12), and RCd (lenalidomide, ifosfamide and dexamethasone, n = 7). Among 19 patients received 1 cycle of treatment 3 patients achieved complete remission (CR), 3 patients achieved very good partial remission (VGPR), 10 patients achieved partial remission (PR) and 3 patients in stable disease (SD) with an overall response rate (ORR = CR + VGPR + PR) of 84%; their ORR rate was 89% after 2 cycles of treatment. In the early stage of treatment, the renal function was improved in 4 out of 5 patients with renal dysfunction. And the common adverse reactions were hematologic toxicity in 4 patients, 1 degree rash in 5 patients, and gastrointestinal side effects in 4 patients. The lenalidomide plus dexamethasone regimen has a good anti-multiple myeloma effect, which can control the disease rapidly and overcome the multidrug resistance in MM, improving the poor prognosis with renal dysfunction, and showing high remission rate in the patients exposed to bortezomib with low toxicity.

A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity.

Multiple myeloma is incurable and invariably becomes resistant to chemotherapy. Although the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in contributing to multiple myeloma progression, angiogenesis, and resistance to chemotherapy. These effects occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIF), and targeting HIFs can have anticancer effects in both solid and hematologic malignancies. Here, it was found that in most myeloma cell lines tested, HIF1α, but not HIF2α expression was oxygen dependent, and this could be explained by the differential expression of the regulatory prolyl hydroxylase isoforms. The anti-multiple myeloma effects of a sequence-specific DNA-binding pyrrole-imidazole (Py-Im) polyamide (HIF-PA), which disrupts the HIF heterodimer from binding to its cognate DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and binds specific regions of DNA with an affinity comparable with that of HIFs. Most of the multiple myeloma cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could overcome this resistance in vitro. Using xenograft models, it was determined that HIF-PA significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a rationale for targeting the adaptive cellular hypoxic response of the O2-dependent activation of HIFα using polyamides. Py-Im polyamides target and disrupt the adaptive hypoxic responses in multiple myeloma cells that may have clinical significance as a therapeutic strategy to treat myeloma engrafted in the bone marrow microenvironment.

Bortezomib and IL-12 produce synergetic anti-multiple myeloma effects with reduced toxicity to natural killer cells

The aim of this study was to examine the hypothesis that a combination of proteasome inhibition by bortezomib and immune therapy with interleukin-12 (IL-12) can produce enhanced antitumor efficacy relative to the effects of either of these agents alone. A mouse xenograft model of myeloma was developed. The mice were randomly divided into saline control (NS), IL-12 (0.4 µg/animal; intraperitoneal), bortezomib (0.75 mg/kg; intravenous), and bortezomib+IL-12 groups. Effects of treatments on tumor growth were assessed by before and after treatment comparisons and group comparisons. The effects of various treatments on the number of peripheral blood lymphocytes and natural killer (NK) cells were assessed by complete blood count and flow cytometry analysis. The cell-killing function of NK cells in splenocytes was evaluated using the lactate dehydrogenase release assay. IL-12 treatment alone produced a mild decrease in tumor volume compared with control (P>0.05). Bortezomib alone resulted in substantial inhibition of tumor growth at varying time points, reaching ~65 and ~60% reduction in tumor volume after 15 and 21 days of therapy, respectively. At the same time points, the combination therapy produced ~75 and ~84% decreases in tumor growth, respectively, which were significantly greater than the reduction produced by bortezomib monotherapy. Tumors resumed growth upon termination of bortezomib treatment at 2 weeks, although the tumor volume was still significantly smaller than that in the time-matched NS and IL-12 animals. This rebound of tumor growth was completely prevented with the combination therapy, and tumor volume continued to decrease throughout the time course. The percentage and total number of NK cells were significantly decreased after bortezomib monotherapy and combination therapy; however, they remained unaltered after IL-12 treatment compared with no treatment. Further, combination therapy significantly restored the bortezomib-induced functional impairment of the cell-killing capability of NK cells, relative to bortezomib alone. We conclude that the bortezomib-IL-12 combination therapy offers superior antitumor efficacy over monotherapy with either bortezomib or IL-12 in a mouse model of myeloma. Restoration of bortezomib-induced functional impairment of NK cells by IL-12 may be a mechanism for the synergetic effects of the two agents. Therefore, a combination of the two agents may represent a more rational therapeutic approach for myeloma.

Multiple myeloma

Nearly all patients with multiple myeloma will eventually relapse; and, thus, it is critical to identify new treatments that increase therapeutic options for these patients. This review highlights the newest approaches with already approved drugs for treating this common B-cell malignancy. Most patients with multiple myeloma in both the frontline and relapsed/refractory settings are now treated with a combination of dexamethasone with the proteasome inhibitor bortezomib and/or an immunomodulatory agent thalidomide or lenalidomide. However, alkylating agents including melphalan, cyclophosphamide and most recently bendamustine as well as anthracyclines, especially the pegylated liposomal doxorubicin, have shown high response rates and prolonged remissions when combined with these agents. There are emerging data showing the importance of maintenance therapy especially with lenalidomide. Because of the marked improvement in survival of multiple myeloma during the past decade, there has been a renewed emphasis on developing therapies that are not only effective but also well tolerated. Alternative dosing, scheduling and routes of administration of already approved drugs have proven effective in accomplishing these goals. The availability of drugs with different mechanisms that produce anti-multiple myeloma effects and also show synergistic effects has paved the way for more effective and safer combinations and led to multiple myeloma patients living longer with improved quality of lives.