A sequential cohort study evaluating single-agent KappaMab and KappaMab combined with lenalidomide and low-dose dexamethasone in relapsed and/or refractory kappa light chain-restricted multiple myeloma (AMaRC 01-16)

KappaMab (KM; formerly MDX-1097) is a monoclonal antibody specific for the kappa myeloma antigen (KMA), a cell-surface antigen expressed on malignant plasma cells in kappa-restricted multiple myeloma (κMM), some lymphomas, occasional tonsillar B cells and in vitro activated B cells, but not on normal B cells in bone marrow. Phase I/IIa studies of single-agent KM confirmed a favourable toxicity profile and evidence of anti-myeloma activity. Ex-vivo studies demonstrating upregulation of KMA by lenalidomide, and enhanced effector-cell cytotoxicity provided the rationale for this phase IIb study where KM or KM in combination with lenalidomide and dexamethasone (KM-Rd) was administered in relapsed, refractory κMM patients. In addition, outcomes for a real-world matched case–control cohort from the Australian and New Zealand Myeloma and Related Diseases Registry (MRDR) who received Rd were compared to the KM-Rd cohort. KM-Rd demonstrated an overall response rate of 82.5% which compared favourably to the Rd-MRDR cohort of 45.1%. Both single-agent KM and KM-Rd regimens were well tolerated, with the KM-Rd safety profile similar to patients given only Rd in other clinical settings. Based on the excellent safety profile and significant efficacy, further clinical trials escalating the KM dose and pairing KM with other standard-of-care treatments are planned.     

Humanized anti-HM1.24 antibody mediates myeloma cell cytotoxicity that is enhanced by cytokine stimulation of effector cells.

To develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1kappa), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16(+) cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16(+) cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.     

Downregulation of BCL2 by AT‐101 enhances the antileukaemic effect of lenalidomide both by an immune dependant and independent manner

Over‐expression of anti‐apoptotic BCL2 has been reported in chronic lymphocytic leukaemia (CLL), but targeting BCL2 alone did not yield appreciable clinical results. However, it was demonstrated that BCL2 inhibitors enhanced the clinical efficacy of chemo and immunotherapeutics. Lenalidomide, an immunomodulator, is clinically effective in CLL and can enhance the anti‐CLL effects of CD20 targeting monoclonal antibody, rituximab. Here, we investigated the mechanism of immune‐directed killing of lenalidomide in CLL and evaluated if concurrent targeting of CD20 and BCL2 can enhance this effect. In vitro treatment with lenalidomide enhanced the antibody‐mediated cellular cytotoxicity (ADCC) directed by rituximab in autologous leukaemic cells. Furthermore, peripheral blood mononuclear cells obtained from patients after treatment with lenalidomide and rituximab showed increased ADCC in vitro versus control (pre‐treatment sample). This effect was further enhanced with pre‐treatment of tumour cells with AT‐101 (a BH3 mimetic that functions as BCL2 antagonist). Our data suggest that AT‐101 in combination with lenalidomide can potentially be an effective therapeutic regimen for CLL.     

Pomalidomide in combination with dexamethasone results in synergistic anti‐tumour responses in pre‐clinical models of lenalidomide‐resistant multiple myeloma

Pomalidomide is an IMiD^® immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, 2004 ; San Miguel et al, 2013; Richardson et al, 2014; Scott, 2014 ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti‐proliferative and pro‐apoptotic activity in both lenalidomide‐sensitive and lenalidomide‐resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single‐agent treatment in xenografts of lenalidomide‐resistant H929 R10‐1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide‐sensitive H929 MM cell lines, were also observed in PomDex‐treated lenalidomide‐resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide‐sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex‐treated samples, highlighted by the modulation of pro‐apoptotic pathways in lenalidomide‐resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide‐resistant setting.     

Individual myeloma‐specific T‐cell clones eliminate tumour cells and correlate with clinical outcomes in patients with multiple myeloma

Despite novel treatment strategies, multiple myeloma (MM) remains an incurable disease with low immunogenicity and multiple immune defects. We developed an ex vivo strategy for inducing myeloma‐specific cytotoxic T lymphocytes (CTLs) and demonstrate the possibility of identification and long‐term in vivo monitoring of individual myeloma‐specific T‐cell clones using the most sensitive clonotypic assay that is able to detect low frequencies of T‐cell clones (1 clonotypic cell in 10⁶ cells). Ten patients with MM were examined for the presence of tumour‐reactive T cells using dendritic cells loaded with autologous tumour cells. All patients had detectable myeloma‐reactive T cells in vitro. Expanded myeloma‐reactive T cells demonstrated specific cytotoxic effects against autologous tumour cells in vitro (median 39·6% at an effector:target ratio of 40:1). The clonality of myeloma‐specific T cells was studied with a clonotypic assay, which demonstrated both oligoclonal and monoclonal populations of myeloma‐specific T cells. CD8⁺ CTLs were the most immunodominant myeloma‐specific T‐cell clones and clinical responses were closely associated with the in vivo expansion and long‐term persistence of individual CD8⁺ T‐cell clones, usually at very low frequencies (10^?3–10^?6). We conclude that the clonotypic assay is the most sensitive tool for immunomonitoring of low‐frequency T cells.     

A novel multi‐epitope vaccine from MMSA‐1 and DKK1 for multiple myeloma immunotherapy

The identification of novel tumour‐associated antigens is urgently needed to improve the efficacy of immunotherapy for multiple myeloma (MM). In this study, we identified a membrane protein MMSA‐1 (multiple myeloma special antigen‐1) that was specifically expressed in MM and exhibited significantly positive correlation with MM. We then identified HLA‐A*0201‐restricted MMSA‐1 epitopes and tested their cytotoxic T lymphocyte (CTL) response. The MMSA‐1 epitope SLSLLTIYV vaccine was shown to induce an obvious CTL response in vitro. To improve the immunotherapy, we constructed a multi‐epitope peptide vaccine by combining epitopes derived from MMSA‐1 and Dickkopf‐1 (DKK1). The effector T cells induced by multi‐epitope peptide vaccine‐loaded dendritic cells lysed U266 cells more effectively than MMSA‐1/DKK1 single‐epitope vaccine. In myeloma‐bearing severe combined immunodeficient mice, the multi‐epitope vaccine improved the survival rate significantly compared with single‐epitope vaccine. Consistently, multi‐epitope vaccine decreased the tumour volume greatly and alleviated bone destruction. The frequencies of CD4⁺ and CD8⁺ T cells was significantly increased in mouse blood induced by the multi‐epitope vaccine, indicating that it inhibits myeloma growth by changing T cell subsets and alleviating immune paralysis. This study identified a novel peptide from MMSA‐1 and the multi‐epitope vaccine will be used to establish appropriate individualized therapy for MM.     

Inhibitory effect of baicalein on IL‐6‐mediated signaling cascades in human myeloma cells

Interleukin‐6 (IL‐6) is an important growth factor for myeloma cells. IL‐6 promotes the survival and proliferation of multiple myeloma (MM) cells through the phosphorylated proteins, including STAT3, MAPK, and Akt. Chemical components that suppress the signaling proteins’ phosphorylation have a potential role for MM therapy. We recently identified that baicalein, a component of Scutellaria radix, suppressed proliferation and induced apoptosis of myeloma cells by blocking IκB‐α degradation followed by down‐regulating IL‐6 and XIAP gene expression. In the present study of four myeloma cell lines, namely U266, NOP2, AMO1, and ILKM2, we demonstrated that baicalein not only inhibited IL‐6‐mediated phosphorylation of signaling proteins, such as Jak, STAT3, MAPK, and Akt, but also inhibited the expression of their target genes, such as bcl‐xl. Finally, baicalein facilitated myeloma cell proliferation inhibited by dexamethasone. In contrast, baicalin, another major flavonoid derived from Scutellaria radix, had no significant effect on IL‐6‐mediated protein phosphorylation. Baicalein had no effect on Akt phosphorylation induced by the insulin‐like growth factor‐1 (IGF‐1) in NOP2 cells. Compared with PD98059, an MAPK inhibitor, baicalein exhibited a stronger inhibitory effect on Erk_1/2 phosphorylation. Our results demonstrate that baicalein is a potent inhibitor of protein phosphorylation induced by IL‐6, and thus may be a useful agent for the treatment of MM.     

Preclinical anti‐myeloma activity of the novel HDAC‐inhibitor JNJ‐26481585

Pharmacological inhibitors of histone deacetylases (HDACs) are currently being developed and tested as anti‐cancer agents and may be useful to enhance the therapeutic efficiency of established anti‐myeloma treatments. This study preclinically evaluated the effects of the ‘second generation’ pan‐HDAC inhibitor JNJ‐26481585 on human multiple myeloma (MM) cells from established cell lines and primary MM samples (n = 42). Molecular responses in both groups of MM cells included histone acetylation, a shift in Bcl2‐family members towards proapoptotic bias, attenuation of growth and survival pathway activity and Hsp72 induction. Mcl‐1 depletion and Hsp72 induction were the most reliable features observed in JNJ‐26481585‐treated primary MM samples. The drug alone effectively induced myeloma cell death at low nanomolar concentrations. In vitro combination of JNJ‐26481585 with anti‐myeloma therapeutic agents generally resulted In effects close to additivity. In view of the favourable activity of this novel HDAC‐inhibitor towards primary myeloma cells further evaluation in a clinical setting is warranted.     

Serum concentrations of DKK‐1 decrease in patients with multiple myeloma responding to anti‐myeloma treatment

Lytic bone destruction is a hallmark of multiple myeloma (MM) and is because of an uncoupling of bone remodeling. Secretion of Dickkopf (DKK)‐1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. In this study, the effect of different treatment regimens for MM on serum DKK‐1 was evaluated and correlated with the response to treatment in 101 myeloma patients receiving bortezomib, thalidomide, lenalidomide, adriamycin and dexamethasone (AD) or high‐dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT). At baseline, myeloma patients had increased serum DKK‐1 as compared with patients with MGUS (mean 3786 pg/mL vs. 1993 pg/mL). There was no difference between previously untreated MM patients and patients at relapse. A significant decrease of DKK‐1 after therapy was seen in the following groups: Bortezomib (4059 pg/mL vs. 1862 pg/mL, P = 0.016), lenalidomide (11837 pg/mL vs. 4374 pg/mL, P = 0.039), AD (1668 pg/mL vs. 1241 pg/mL, P = 0.016), and AD + HDCT + ASCT (2446 pg/mL vs. 1082 pg/mL, P = 0.001). Thalidomide led to a non‐significant decrease in DKK‐1 (1705 pg/mL vs. 1269 pg/mL, P = 0.081). Within all groups, a significant decrease of DKK‐1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non‐responders. We show for the first time that serum DKK‐1 levels decrease in myeloma patients responding to treatment, irrespective of the regimen chosen. These data suggest that myeloma cells are the main source of circulating DKK‐1 protein and provide a framework for clinical trials on anti‐DKK‐1 treatment in MM.     

A CD138‐independent strategy to detect minimal residual disease and circulating tumour cells in multiple myeloma

CD138 (also termed SDC1) has been the gold‐standard surface marker to detect multiple myeloma (MM) cells for decades; however, drug‐resistant residual and circulating MM cells were shown to have lower expression of this marker. In this study, we have shown that residual MM cells following bortezomib treatment are hypoxic. This combination of drug exposure and hypoxia down‐regulates their CD138 expression, thereby making this marker unsuitable for detecting residual or other hypoxic MM cells, such as circulating tumour cells, in MM. Hence, we developed an alternative biomarker set which detects myeloma cells independent of their hypoxic and CD138 expression status in vitro, in vivo and in primary MM patients. The new markers were able to identify a clonal CD138‐negative population as minimal residual disease in the bone marrow and peripheral blood of MM patients. Further investigation to characterize the role of this population as a prognostic marker in MM is warranted.     

NK cell dysfunction with down‐regulated CD16 and up‐regulated CD56 molecules in patients with esophageal squamous cell carcinoma

NK cells can be divided into two subsets, CD56^dim and CD56^bright NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56^dim or CD56^bright NK cells gated on CD56(+)CD3(–) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab‐mediated antibody‐dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)‐expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56^dim and CD56^bright NK cells between ESCC patients and healthy donors, down‐regulated CD16 and up‐regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab‐mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down‐regulated CD16 and up‐regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF‐beta1 partially contributed to down‐regulation of CD16 on NK cells. Down‐regulated CD16 and up‐regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.     

Identification of human leucocyte antigen (HLA)‐A*0201‐restricted cytotoxic T lymphocyte epitopes derived from HLA‐DOβ as a novel target for multiple myeloma

Despite the recent development of effective therapeutic agents against multiple myeloma (MM), new therapeutic approaches, including immunotherapies, remain to be developed. Here we identified novel human leucocyte antigen (HLA)‐A*0201 (HLA‐A2)‐restricted cytotoxic T lymphocyte (CTL) epitopes from a B cell specific molecule HLA‐DOβ (DOB) as a potential target for MM. By DNA microarray analysis, the HLA‐DOB expression in MM cells was significantly higher than that in normal plasma cells. Twenty‐five peptides were predicted to bind to HLA‐A2 from the amino acid sequence of HLA‐DOB. When screened for the immunogenicity in HLA‐A2‐transgenic mice immunized with HLA‐DOB cDNA, 4 peptides were substantially immunogenic. By mass spectrometry analysis of peptides eluted from HLA‐A2‐immunoprecipitates of MM cell lines, only two epitopes, HLA‐DOB_232–240 (FLLGLIFLL) and HLA‐DOB_185–193 (VMLEMTPEL), were confirmed for their physical presence on cell surface. When healthy donor blood was repeatedly stimulated in vitro with these two peptides and assessed by antigen‐specific γ‐interferon secretion, HLA‐DOB_232–240 was more immunogenic than HLA‐DOB_185–193. Additionally, the HLA‐DOB_232–240‐specific CTLs, but not the HLA‐DOB_185–193‐specific CTLs, displayed an major histocompatibility complex class I‐restricted reactivity against MM cell lines expressing both HLA‐A2 and HLA‐DOB. Taken together, based on the physical presence on tumour cell surface and high immunogenicity, HLA‐DOB_232–240 might be useful for developing a novel immunotherapy against MM.     

Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.     

B‐Lymphocyte stimulator: a new biomarker for multiple myeloma

Multiple myeloma (MM) is a common malignant tumor, characterized by unlimited proliferation of abnormal plasmocytes in bone marrow. Considering the biological function of B‐Lymphocyte stimulator (BLyS) and its receptors in B cell, we examined BLyS and its receptors expression in MM cells. Our studies confirmed that BLyS and its receptors are expressed in MM cells, including KM3, CZ‐1, and primary MM cells, playing an important role in the survival and proliferation of MM cells. Additionally, we provide evidence that BLyS protein is located in the MM cell plasma membrane. We also found that IFN‐γ and IL‐6 can induce BLyS expression on MM cells, while after the treatment of BAY11‐7082, an IkB‐α phosphorylation inhibitor, IFN‐γ induced up regulation of BLyS was completely inhibited, suggesting that nuclear factor κB (NF‐κB) might be involved in the mechanism of the regulation of BLyS levels in response to cytokines. Finally, linear correlation analysis of the Lactate Dehydrogenase concentration and beta 2‐microglobulin level with BLyS, and expressions of BLyS mRNA in MM patients revealed a significant correlation between them (P < 0.01 in all case), showing that BLyS could be a biomarker for the diagnosis and treatment of MM.     

Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor,induces pleiotropic anti‐myeloma activity in vitro and in vivo

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine‐induced osteoclast differentiation more potently (9‐ to 10‐fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G₀‐G₁ cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti‐MM therapies. Significant tumour growth reduction in AS703026‐ vs. vehicle‐treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC‐induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti‐MM agents, to improve patient outcome.     

The humanized CD40 antibody SGN-40 demonstrates pre-clinical activity that is enhanced by lenalidomide in chronic lymphocytic leukaemia

Antibody-based therapies, such as rituximab and alemtuzumab, have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy.     

Unique anti‐myeloma activity by thiazolidine‐2,4‐dione compounds with Pim inhibiting activity

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine‐2,4‐dione‐family compounds SMI‐16a and SMI‐4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX‐6258 and PIM447. SMI‐16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony‐forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination‐independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI‐16a mitigated the PIM2 protein increase and cooperatively enhanced anti‐MM effects in combination with carfilzomib. Collectively, the thiazolidine‐2,4‐dione‐family compounds SMI‐16a and SMI‐4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.     

Preclinical activity of a novel multiple tyrosine kinase and aurora kinase inhibitor,ENMD‐2076, against multiple myeloma

ENMD‐2076 is a novel, orally‐active molecule that has been shown to have significant activity against aurora and multiple receptor tyrosine kinases. We investigated the activity of ENMD‐2076 against multiple myeloma (MM) cells in vitro and in vivo. ENMD‐2076 showed significant cytotoxicity against MM cell lines and primary cells, with minimal cytotoxicity to haematopoietic progenitors. ENMD‐2076 inhibited the phosphoinositide 3‐kinase/AKT pathway and downregulated survivin and X‐linked inhibitor of apoptosis as early as 6 h after treatment. With longer treatment (24–48 h), ENMD‐2076 also inhibited aurora A and B kinases, and induced G₂/M cell cycle arrest. In non‐obese diabetic/severe combined immunodeficient mice implanted with H929 human plasmacytoma xenografts, oral treatment with ENMD‐2076 (50, 100, 200 mg/kg per day) resulted in a dose‐dependent inhibition of tumour growth. Immunohistochemical staining of excised tumours showed significant reduction in phospho‐Histone 3 (pH3), Ki‐67, and angiogenesis, and also a significant increase in cleaved caspase‐3 at all dose levels compared to tumours from vehicle‐treated mice. In addition, a significant reduction in p‐FGFR3 was observed on Western blot. ENMD‐2076 shows significant activity against MM cells in vitro and in vivo, and acts on several pathways important for myeloma cell growth and survival. These results provide preclinical rationale for clinical investigation of ENMD‐2076 in MM.