Cooperative Supression of MEIS2 Mediates Dexamethasone Enhancement of Lenalidomide Killing in Myeloma Cells

Abstract Dexamethasone (Dex) enhances the clinical response to most therapies in multiple myeloma, the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide in particular. As a steroid, Dex may modulate the therapeutic response by acting on multiple cell types in vivo. Whether and how Dex enhances the tumor intrinsic clinical response to IMiDs is not well understood. To address this important question, we focus on the role of Dex in modulating the proximal events in IMiD signaling that lead to the killing of myeloma cells. Cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. MEIS2 is a homeobox transcription factor of the mammalian myeloid ecotropic insertion site (MEIS) family, which plays a pivotal role in development and leukemogenesis. Recent crystal structure studies in conjunction with biochemical screen have identified MEIS2 as an endogenous substrate of CRBN. IMiDs apparently bind CRBN and block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for degradation. This then leads to loss of IRF4, a target of IKZF1/3, necessary for myeloma survival. This model posits that MEIS2 inhibits the function of CRBN in myeloma cells. However, as all the studies of MEIS2 were performed heterologous cell lines, evidence that supports a function of MEIS2 in myeloma cells is lacking. We now showed by RNA-seq and protein analysis that MEIS2 was absent in normal bone marrow plasma cells (BMPC), but highly expressed in freshly isolated primary bone marrow myeloma cells (BMMC)s at both RNA and protein levels. We further showed that MEIS2 was regulated by the cell cycle: reduced in G1 arrest and restored in S phase in human myeloma cell lines (HMCL)s as well as primary BMMCs. Depletion of MEIS2 markedly enhanced lenalidomide killing, and overexpression of MEI2 attenuated lenalidomide killing of HMCL MM1.S. These results provide the first evidence that MEIS2 is aberrantly expressed in primary myeloma cells and acts within myeloma cells to inhibit IMiD killing. To investigate if Dex enhances the myeloma intrinsic response to IMiDs, we first determined that lenalidomide and pomolidomide induced late G1 arrest within 24 hours, as evidenced by the repression of cyclin A, the increases in p21 and p27 proteins, and the reduction of myeloma cells in S phase by BrdU labeling. Kinetic studies further indicate that induction of G1 arrest precedes apoptosis, detectable at 72 hours in MM1.S and CAG cells. These data suggest that IMiDs preferentially kill myeloma cells arrested in G1. Dex killed CAG and MM1.S cells marginally at 10 nM and killing was not greater at 100 nM. However, concurrent addition of Dex enhanced lenalidomide killing, more prominently in CAG cells then in MM1.S cells. Addition of Dex at these concentrations to CAG cells after 48 hours of exposure to lenalidomide leads to synergistic and greater killing in 24 hours in a dose-dependent manner. These data indicate that Dex enhances lenalidomide killing, preferentially in G1 arrest. To investigate the mechanism by which Dex enhances lenalidomide killing, we found that Dex did not regulate MEIS2 or CRBN protein expression in CAG cells, in line with its inability to significantly kill myeloma cells. Sequential addition of lenalidomide and Dex led to a > 2-fold reduction in the MEIS2 protein, which corresponds to a > 2-fold reduction in the MEIS2/CRBN ratio and synergistic killing. Concurrent addition of Dex and lenalidomide also resulted in a 2-fold reduction of the MEIS2 protein and the MEIS2/CRBN ratio, but it required 72 hours of the presence of Dex instead of 24 hours. In summary, our study demonstrates, for the first time, that MEIS2 is expressed in primary myeloma cells and inhibits IMiDs killing. Dex enhances IMiD killing of myeloma cells through cooperative suppression of MEIS2 at proximal IMiD signaling. These findings suggest a novel mechanism by which Dex enhances the anti-myeloma activity of IMiD, which has important implication for myeloma therapy. Disclosures Rossi: Celgene: Consultancy, Speakers Bureau; Onyx: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Janssen: Speakers Bureau. Niesvizky:Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Research Funding, Speakers Bureau.

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Lenalidomide Targets Myeloma Cells Preferentially During Prolonged Early G1 Arrest but Not Synchronization Into S Phase by Selective and Reversible Inhibition of CDK4/CDK6 through Loss of IRF-4

Blood ◽

10.1182/blood.v116.21.449.449 ◽

2010 ◽

Vol 116 (21) ◽

pp. 449-449 ◽

Cited By ~ 1

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

David Jayabalan ◽

Mohamad Hussein ◽

Sophia Randolph ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Proteasome Inhibitor ◽

Research Funding ◽

Phase 1 ◽

Proteasome Inhibitors ◽

S Phase ◽

G1 Arrest ◽

Myeloma Cells ◽

Sequential Combination

Abstract Abstract 449 Dysregulation of cyclin-dependent kinase (CDK)4 and CDK6 is common in human cancer and precedes unrestrained proliferation of tumor cells in multiple myeloma (MM) patients, especially during refractory relapse. MM remains incurable due to the eventual development of drug resistance despite initial response to two main lines of therapy with proteasome inhibitors and immunomodulatory drugs. Therapeutic strategies that both control the cell cycle and enhance cytotoxic killing are thus urgently needed in MM. Although targeting the cell cycle in cancer therapy has only been modestly successful because broad-spectrum CDK inhibitors lack specificity and are highly toxic, we have recently developed such a therapy by selective inhibition of CDK4/CDK6 in sequential combination with proteasome inhibitors. Using PD 0332991, the only known selective inhibitor of CDK4/CDK6 that is also potent, reversible and bioavailable, we have demonstrated that 1) induction of prolonged early G1 arrest by inhibition of CDK4/CDK6 markedly enhances the killing of primary BM myeloma cells by proteasome inhibitors despite stromal protection, and 2) release from the G1 block upon PD 0332991 withdraw leads to synchronous progression to S phase, which further augments cytotoxic killing of MM cells. To optimize targeting CDK4/CDK6 in MM, we have investigated lenalidomide as an alternative cytotoxic partner by first determining its cell cycle targeting specificity, taking advantage of the exceptional precision and efficiency with which PD 0332991 induces early G1 arrest and cell cycle synchronization. We show by simultaneous analyses of BrdU pulse labeling and DNA content per cell that lenalidomide preferentially targets MM cells following prolonged early G1 arrest by PD 0332991 pretreatment for 24 hours (twice the time needed to induce G1 arrest in MM cells), but not those synchronized into S phase after release from the G1 block. This is distinct from proteasome inhibitors (bortezomib, carfilzomib and ONX-0921), which preferentially target MM cells synchronized into S phase over those arrested in G1. MM cells in G2/M appear to be less sensitive to both proteasome inhibitors and lenalidomide. However, these cells are rendered sensitive to these compounds upon cell cycle reentry through inhibition of CDK4/CDK6 and induction of early G1 arrest. Time course studies of DNA replication further reveal that lenalidomide alone (3 uM, daily) induces G1 arrest by 48 hours, which precedes evidence of apoptosis and reduction of viable cells at 72 hours. While the magnitude of G1 arrest induced by lenalidomide is dose-dependent (1-50 uM), the timing of cytotoxic killing does not vary. Prior induction of prolonged early G1 arrest by PD 0332991 (24 hours) enhances (> 5-fold) and also accelerates lenalidomide killing by at least 24 hours, leading to eradication of some MM cell lines. This acceleration and enhancement of lenalidomide killing appears to be mediated by synergistic reduction of IRF-4, as we have found in cell cycle enhancement of proteasome inhibitor killing. Most importantly, acceleration of early G1 arrest by inhibition of CDK4/CDK6 in primary bone marrow myeloma cells enhances lenalidomide killing in the presence of bone marrow stromal cells. Thus, the immunomodulatory compound lenalidomide induces G1 arrest and is cytotoxic for myeloma cells directly and preferentially in G1, in contrast to proteasome inhibitors, which preferentially target MM cells in S phase. Induction of prolonged early G1 arrest accelerates and enhances subsequent lenalidomide killing, which appears to be mediated by loss of IRF-4 in common with cell cycle enhancement of proteasome inhibitor killing. To implement targeting CDK4/CDK6 in combination therapy, a multi-center phase 1/2 clinical trial targeting CDK4/6 with PD 0332991 in sequential combination with bortezomib and dexamethasone in relapsed refractory MM is in progress. Data from the phase 1 portion indicate that PD 0332991 is well tolerated, and directly and completely inhibits CDK4/CDK6 and the cell cycle in tumor cells in MM patients with promising clinical efficacy. Given the known clinical efficacy of lenalidomide in MM, our findings suggest lenalidomide as an attractive cytotoxic partner for targeting CDK4/CDK6 in sequential combination therapy to both control tumor expansion and enhance tumor killing in the treatment of MM. Disclosures: Off Label Use: PD 0332991 is a cell cycle CDK4/CDK6 inhibitor. Hussein:Celgene: Employment. Randolph:pfizer: Employment, Equity Ownership. Niesvizky: Celgene: Consultancy, Research Funding, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau; Onyx: Consultancy, Research Funding, Speakers Bureau.

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Induction of sequential G1 arrest and synchronous S phase entry by reversible CDK4/CDK6 inhibition sensitizes myeloma cells for cytotoxic killing through loss of IRF-4.

Blood ◽

10.1182/blood.v114.22.299.299 ◽

2009 ◽

Vol 114 (22) ◽

pp. 299-299

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

Scott Ely ◽

David S Jayabalan ◽

Isan Chen ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Research Funding ◽

Cell Cycle Progression ◽

Specific Inhibitor ◽

S Phase ◽

G1 Arrest ◽

Myeloma Cells ◽

Cycle Dependent ◽

Phase Entry

Abstract Abstract 299 Dysregulation of the cell cycle is a hallmark of cancer. However, targeting the cell cycle in cancer therapy has only been modestly successful since broad-spectrum cyclindependent kinase (CDK) inhibitors lack specificity and are highly toxic. The critical importance of controlling CDK4/CDK6 in cancer treatment is further exemplified by recent evidence of prominent CDK4/CDK6 dysregulation in human cancers, including breast cancer, metastatic lung adenocarcinoma, glioblastoma, mantle cell lymphoma and multiple myeloma (MM). To advance mechanism-based targeting of the cell cycle in cancer, we have developed a novel strategy that both inhibits cell cycle progression and enhances cytotoxic killing in tumor cells using PD 0332991(PD), the only known CDK4/CDK6-specific inhibitor that is also reversible, potent and orally bioavailable. We demonstrated by BrdU pulse-labeling that inhibition of CDK4/CDK6 with PD in primary bone marrow (BM) myeloma cells and human myeloma cell lines (HMCL) (IC50 60nM) leads to a complete early G1 arrest in the absence of apoptosis and upon release of the G1 block, synchronous cell cycle progression to S phase. Furthermore, prolonged early G1 arrest enhances cytotoxic killing of MM cells by diverse clinically relevant drugs at low dose, including bortezomib, carfilzomib (PR-171) and dexamethasone, and this is dramatically augmented during synchronous S phase entry. The enhancement of cytotoxic killing in either G1 arrest or synchronous S phase entry is sustained in the presence of BM stromal cells. This killing is caspase-dependent and triggered by the loss of mitochondrial outer membrane potential and activation of the intrinsic apoptosis pathway. Time course studies of cell cycle-specific gene expression by expression profiling, quantitative real time RT-PCR and immunoblotting further revealed that the expression of IRF-4, essential for normal plasma cell differentiation and myeloma cell survival, is strictly cell cycle-dependent: elevated in G1 and markedly declined in S phase. The IRF-4 protein is also markedly reduced (50%) by bortezomib treatment, resulting in a combined 5-fold reduction in S phase. This suggests that differential enhancement of cytotoxic killing in G1 arrest and S phase is mediated by cell cycle-dependent IRF-4 expression. Indeed, shRNA interference confirms that by antagonizing mitochondrial depolarization, IRF-4 is required to protect myeloma cells from cell cycle-dependent enhancement of bortezomib killing. By timely administration and discontinuation of PD treatment, we have further demonstrated in a human MM 1.S. xenograft myeloma model that it is feasible to induce sequential G1 arrest and synchronous S phase in vivo. This leads to synergistic tumor suppression through amplification of bortezomib killing of myeloma cells, but not normal BM cells. As PD is orally bio-available, specific and low in toxicity, our novel strategy has been implemented in the first phase I/II multi-center clinical trial targeting CDK4/CDK6 with PD in combination with bortezomib and dexamethasone in MM. Preliminary bone marrow immunohistochemistry demonstrates PD preferentially and completely inhibits CDK4/CDK6-specific phosphorylaton of Rb and DNA replication in tumor cells, but not other bone marrow cells in all patients. One patient achieved VGPR (12.5%) while 1 patient each achieved MR and SD respectively for an ORR 25% (Niesvizky et al, submitted). Collectively, our preclinical and clinical data indicate, for the fist time, that selective targeting of CDK4/CDK6 in combination therapy is a promising mechanism-based therapy for MM and potentially other cancers. Disclosures: Off Label Use: PD 0332991 is going to be used as a CDK4/6-specific inhibitor.. Chen:Pfizer, Inc.: Employment, Equity Ownership. Wilner:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Millenium: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Seattle Genetics, Inc: Research Funding; Proteolix: Research Funding, data monitoring committee. Chen-Kiang:Pfizer Inc.: Research Funding.

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KRN5500, a Spicamycin Derivative, Exerts Anti-Myeloma Effects through Impairing Both Myeloma Cells and Osteoclasts.

Blood ◽

10.1182/blood.v112.11.1669.1669 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1669-1669

Author(s):

Hirokazu Miki ◽

Shuji Ozaki ◽

Osamu Tanaka ◽

Shingen Nakamura ◽

Ayako Nakano ◽

...

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Cell Death ◽

Cell Growth ◽

Cell Lines ◽

G1 Arrest ◽

Dependent Manner ◽

Osteolytic Bone Disease ◽

Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by devastating bone destruction due to enhanced bone resorption and suppressed bone formation. Although high-dose chemotherapy and new agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains incurable due to drug resistance mediated by interactions with osteoclasts or stroma cells. Moreover, osteolytic bone disease continues to be a major problem for many patients. Therefore, alternative approaches are necessary to overcome drug resistance and inhibit osteoclasts activity in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan), which potently inhibits protein synthesis and induces cell death in human tumor cell lines. Phase I studies of KRN5500 in patients with solid tumors such as colon cancer and gastric cancer showed acceptable toxicity with Cmax values of 1000––3000 nM. In this study, we investigated the effects of KRN5500 against MM cells and osteoclasts in vitro and in vivo. MM cell lines such as RPMI 8226, MM.1S, INA-6, KMS12-BM, UTMC-2, TSPC-1, and OPC were incubated with various concentrations of KRN5500 for 3 days. Cell proliferation assay showed marked inhibition of cell growth with G1 arrest in these MM cells (IC50: 4–100 nM). KRN5500 (100 nM) also induced 30–90% of cell death in primary MM cells (n=7). Annexin V/propidium iodide staining showed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Western blot analysis confirmed activation of caspase-8, -9, and −3, cleavage of poly (ADP-ribose) polymerase (PARP), and down-regulation of Mcl-1. We next examined the effect of KRN5500 against MM cell lines and primary MM cells in the presence of bone marrow stroma cells and osteoclasts. Co-culture of these cells enhanced viability of MM cells; however, KRN5500 still induced strong cytotoxicity to MM cells. Of interest, KRN5500 specifically mediated apoptosis in osteoclasts but not stroma cells as assessed by TUNEL staining. More than 90% of osteoclasts were killed even at a low concentration of KRN5500 (20 nM). Finally, we evaluated the effect of KRN5500 against MM cells and osteoclasts in vivo. Two xenograft models were established in SCID mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells into subcutaneously implanted rabbit bones (SCID-rab model). These mice were treated with intraperitoneal injection of KRN5500 (5 mg/kg/dose) or saline thrice a week for 3 weeks after tumor development. In a subcutaneous tumor model, KRN5500 inhibited the tumor growth compared with control mice (increased tumor size, 232 ± 54% vs 950 ± 422%, p<0.001, n=6 per group). In a SCID-rab model, KRN5500 also inhibited MM cell growth in the bone marrow (increase of serum human sIL6-R derived from INA-6, 134 ± 19% vs 1112 ± 101%, p<0.001, n=5 per group). Notably, the destruction of the rabbit bones was also prevented in the KRN5500-treated mice as evaluated by radiography. Therefore, these results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts and that this unique mechanism of action provides a valuable therapeutic option to improve the prognosis in patients with MM.

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Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽

10.1182/blood.v122.21.275.275 ◽

2013 ◽

Vol 122 (21) ◽

pp. 275-275

Author(s):

Siobhan Glavey ◽

Salomon Manier ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Yuji Mishima ◽

...

Keyword(s):

Bone Marrow ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Myeloma Cells ◽

Sialyl Lewis ◽

And Migration ◽

Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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Unravelling the Complexities of Hedgehog Mediated Signal Transduction in Acute Myeloid Leukaemia and Normal Haematopoiesis

Blood ◽

10.1182/blood.v128.22.2881.2881 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2881-2881

Author(s):

Victoria Campbell ◽

Anuradha Tarafdar ◽

Edwina Dobbin ◽

Gillian A. Horne ◽

Laura Park ◽

...

Keyword(s):

Signal Transduction ◽

Bone Marrow ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Primary Cilia ◽

Hematopoietic Cells ◽

Advisory Committees ◽

Fold Reduction

Abstract Primary cilia regulate hedgehog (Hh) signal transduction; these sensory organelles being present on most mammalian cells. Aberrant Hh activity has been implicated in malignancy with defective primary cilia expression linked to disease. The role the Hh pathway plays in AML has not been fully elucidated and it is unclear whether AML cells express primary cilia. Our aims were (1) to determine the presence of primary cilia and (2) further evaluate the role of Hh signalling in AML. Primary cilia were identified in all AML (n=23), and 20% of normal (n=10) bone marrow trephines (BMTs) by immunocytochemistry (ICC). Primary cilia were not identified in AML cell lines, primitive (n=4) or mature hematopoietic cells (n=6) isolated from peripheral blood, suggesting they are lost once cells migrate from the bone marrow (BM) microenvironment. Despite the heterogeneity of AML, analysis of 76 primary AML samples demonstrated clear evidence of Hh pathway activity with up regulation of SMO (p<0.001), confirmed by ICC (n=8), and Gli-1 (p<0.05) in 55% of cases. Sanger sequencing (n=36) did not identify a mutation in SMO to account for the increased activity, although genomic instability was apparent with a high level of SNPs (mean 4; range 3-8), and silent and missense mutations (mean 1; range 1-3 per sample) identified. Samples with a >5-fold increase in SMO (n=28) showed a >5-fold up regulation of genes associated with chemoresistance and poor survival, including MECOM and FOXM1 (both p=0.0001), ABCC1, HOXA3, HOXA9, TWIST1 and SNAIL1 (all p<0.001), KIT (p<0.01), CD44, MMP2 and DNMT3b (all p<0.05); with a 4-fold reduction in pro-apoptotic genes BAK and BAX. Next Gen sequencing (n=9) confirmed an aggressive phenotype with 67% of patients having a c-Kit mutation and 46% (n=13), stratifying within the high risk category. The BM microenvironment is important in AML. Immunohistochemistry (IHC) on human AML BMTs (n=37) enabled analysis within this unique environment. Proteins associated with poor prognosis and a more aggressive phenotype were up regulated 2-30 fold, including BCL-2 (p<0.0001), CD44 (p<0.001) and Vimentin (p<0.05). SHH was significantly down regulated (p<0.001) within the blast population. Secreted SHH, measured by ELISA, was up regulated suggesting paracrine activity. Impaired post translational modification of SHH was demonstrated with protein located within the nuclei by IHC and ICC. Nuclear expression of SHH was limited to primitive (CD34+) cells and absent from mature (CD14+, CD15+) cells. This correlated with a 20-fold reduction in HHAT the acetyltransferase involved in Hh processing in primitive cells compared to MNCs (p=<0.01). In vitro, SMO inhibition with cyclopamine reduced cell proliferation in myelomonocytic cell lines (Kasumi-1 p<0.05, MOLM-13 p<0.01, MV4-11 p<0.05, OCI-AML3 p<0.001 and THP-1 p<0.01). No change in early or late apoptosis was seen. Cell cycle arrest was demonstrated with an increase in G0-G1 within OCI-AML3 (p<0.05) and a 3-fold reduction in cell division. Cells demonstrated a striking morphological change with increased cytoplasm, granules and loss of nucleoli. Reduction in cell proliferation and morphological changes were also observed when SMO was knocked down using a SMO siRNA. OCI-AML3 cells demonstrated increased expression of CD11b (p<0.001) and CD11c (p<0.0001) by FACS in keeping with a more mature phenotype. Gene expression correlated with a 2-fold reduction in early lineage markers, GATA1, SCL, NAB2 (p<0.05) and 3-4 fold up regulation of genes involved in differentiation and maturation, PU1, GMCSF and GCSF (all p<0.05), and IRF8 (p<0.01). Primary AML samples (n=6) were treated with the clinical grade SMO antagonist, PF-04449913, alone and in combination with cytarabine. Combination treatment caused a significant reduction in proliferation (p<0.05) with an increase in apoptosis. LTCIC showed a trend towards differentiation with a greater number of CFU-M and CFU-G than CFU-GM compared to no drug control (n=4). This is the first report to demonstrate primary cilia on hematopoietic cells, with an increased frequency observed in AML. Their absence when cells migrate from the BM fits with their function and suggests a 'switching off' of the Hh pathway occurs on maturation. The ability of SMO inhibition to cause differentiation, in genetically diverse AML cell lines and primary AML is promising. SMO inhibition should continue to be explored as a potential therapy in AML. Disclosures Dobbin: Almac: Employment. Wheadon:GlaxoSmithKline: Research Funding. Copland:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Amgen: Honoraria; Shire: Honoraria.

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A Novel Putative Mechanism of Anti-Myeloma Activity Targeted Against Heat Shock Protein 27 by Derivative of Artemisinin, Artesunate.

Blood ◽

10.1182/blood.v110.11.1521.1521 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1521-1521

Author(s):

Aki Chizuka ◽

Hideo Uchida ◽

MingJi Xian ◽

Takayuki Shimizu ◽

Yasuo Ikeda ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Cell Lines ◽

Myeloma Cell ◽

Inhibitory Effect ◽

G1 Arrest ◽

Dependent Manner ◽

Myeloma Cells ◽

Hsp 27 ◽

Induction Of Apoptosis

Abstract Recent introduction of molecular targeted drugs such as bortezomib and IMIDs in the clinical settings has achieved the improved treatment outcome of multiple myeloma (MM). However, MM is still an incurable disease and these drugs also possess serious adverse reactions, therefore, safe and more effective therapy should be established. Artesunate (ART) is a semi-synthetic derivative of artemisinin and is widely used for the treatment of malaria as a salvage therapy. Recent in vitro studies showed that ART also has an anti-tumor activity against several cancer cell lines. We thus investigated whether ART could possess anti-myeloma activity and demonstrated that apoptosis of myeloma cells is strongly induced by ART. Interestingly, we also found that this activity is mediated through heat shock protein (Hsp) 27-dependent pathway. Several MM cell lines (IM9, OPM2, RPMI8226, U266) were treated with various concentrations of ART for 48 hours, and MTT assays were performed to assess the anti-myeloma activity. IM9, OPM2, RPMI8226 cells showed the striking reduction of viability (up to 40% at 1μM and up to 90% at 10μM) in a dose- and time-dependent manner, whereas ART has less inhibitory effect on U266 cell line. ART induced G1 arrest of IM9 cells, and apoptosis was confirmed by decreased mitochondrial membrane potential, and flow cytometric analysis using AnnexinV/PI staining. Colony assay showed that ART has no growth inhibitory effect on normal CD34-positive bone marrow cells even at a concentration of 10μM. Immunoblot analyses demonstrated the activation of caspases-3 and -9, and the decreased expression of pro-apoptotic protein Bid. Interestingly, heat shock protein (Hsp) 27 was downregulated in myeloma cell lines (IM9, RPMI8226) which are sensitive to the ART treatment. In contrast, downregulation of Hsp 27 was not observed in U266 cells which are resistant to ART. Other anti-apoptotic Hsps (Hsp70, 90), as well as Bcl-2 family proteins (Bcl-2, Bax, Bad, Bcl-xL), Akt, MDM2, p53 and MAPKs (SAPK/JNK, p38, ERK1/2) were not affected by ART. Quantitative RT-PCR analysis showed that ART did not influence the mRNA expression of Hsp27, suggesting that ART could downregulate the Hsp27 protein at a posttranscriptional or posttranslational levels. Overexpression of Hsp27 cDNA by transfection method in ART-sensitive myeloma cell lines demonstrated that these cell lines acquired resistance to ART and apoptosis was not induced at a concentration which ART is effective to the parental cells. Our preliminary data using knockdown procedure of Hsp27 mRNA by RNAi-expressing lentivirus showed the growth suppression of myeloma cells, which apparently suggested that Hsp27 could confer a critical function in the proliferation of myeloma cells and that downregulation of Hsp27 could result in induction of apoptosis in MM. Hsp27 has an important role in anti-apoptotic effect against various stresses to the cells. Our data implies a novel mechanism that downregulation of Hsp27 by ART could induce misfolding of several client proteins indispensable for the cell growth, which results in the induction of apoptosis in MM cells. In conclusion, ART could become a candidate of safe and effective therapeutic drug for MM, and Hsp 27 might be a potential molecular target for the treatment of MM.

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Pre-Clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Mutiple Myeloma Tumors

Blood ◽

10.1182/blood.v112.11.3667.3667 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3667-3667 ◽

Cited By ~ 1

Author(s):

Seda Zeng ◽

Zhi Hua Li ◽

Marta Chesi ◽

Chungyee Leung-Hagesteijn ◽

Sheng-ben Liang ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Lines ◽

Cell Cycle Progression ◽

Myeloma Cell ◽

S Phase ◽

Causative Role ◽

Cyclin D ◽

Cycle Progression ◽

Myeloma Cells

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

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A Phase I Trial of PD 0332991, a Novel, Orally-Bioavailable CDK4/6-Specific Inhibitor Administered in Combination with Bortezomib and Dexamethasone to Patients with Relapsed and Refractory Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.1877.1877 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1877-1877 ◽

Cited By ~ 2

Author(s):

Ruben Niesvizky ◽

Scott Ely ◽

David S Jayabalan ◽

Megan C. Manco ◽

Seema Singhal ◽

...

Keyword(s):

Cell Cycle ◽

Animal Models ◽

Dose Escalation ◽

Research Funding ◽

Specific Inhibitor ◽

S Phase ◽

Stage Ii ◽

G1 Arrest ◽

Myeloma Cells ◽

Orally Bioavailable

Abstract Abstract 1877 Poster Board I-902 Background: PD 0332991 (PD) a potent orally bioavailable small molecule, is the only known CDK4/CDK6-specific inhibitor. Through selective inhibition of CDK4/6, PD induces G1 cell cycle arrest thereby preventing DNA replication. As PD acts reversibly, it can induce synchronous G1-S progression upon its discontinuation. It has been shown that PD potently induces G1 arrest in primary human bone marrow (BM) myeloma cells/lines in the presence of BM stroma and prevents tumor growth in animal models (IC50, ∼60 nM) (Baughn et al 2006). Moreover, it has been shown that induction of prolonged G1 arrest and synchronous S phase entry by PD profoundly enhances bortezomib (B) and dexamethasone (D) killing of primary BM myeloma cells in vitro and in animal models (Huang et al, unpublished). The synergistic anti-myeloma effect provides a compelling rationale for evaluating two methods of targeting CDK4/6 using PD in combination with B and D: a “concurrent regimen” aims at enhancing B killing during prolonged G1 arrest and a “sequential regimen” to enhance B killing during both G1 arrest and synchronous progression to S phase. Methods: Patients (pts) who had relapsed and refractory myeloma after at least 2 previous treatments and who were Rb positive were eligible. This phase 1 dose escalation study is being conducted to assess the safety, tolerability and pharmacokinetics of the combination including dose limiting toxicities (DLTs). PD was given orally once a day for 3 weeks (21 days) followed by one week (7 days) off (Schedule A), starting on Day 1 of each cycle. B was given during G1 arrest by IV bolus together with 20 mg PO of D on Days 8, 11, 15 and 18 of each cycle (up to a maximum of 10 cycles). The study design followed a 3+3 dose-escalation scheme, with planned doses of PD/B starting from 100 mg/1.0 mg/m2 and escalating up to a maximum dose of 125 mg/1.3 mg/m2, respectively. Alternatively, in case of toxicity, de-escalation was planned to a minimum dose of 50 mg/0.7 mg/m2 for PD/B, respectively. Results: Nine patients were enrolled in Schedule A. Pt characteristics included 90% > SD stage II, 50% > ISS stage II with median β2 M 4.0 (range 1.6–10.5), median serum albumin 3.8 (2.2–4.6), median Hgb 10.5 (8.1–14.4) and median calcium 9.9 (8.7–10.7) values at screening. The median number of prior therapies was 6 (2–10) with 7/9 pts showing progression to their latest prior therapy. One patient achieved VGPR (12.5%) while 1 patient each achieved MR and SD respectively for an ORR 25%. The VGPR and MR were achieved with the lowest dose combination (75 mg/0.7 mg/m2 PD/B). Six pts had progression of disease while on therapy. The most commonly reported adverse events were >Grade 3 reversible uncomplicated cytopenias. PD was absorbed relatively slowly with a median Tmax of 4 hours (range 2–8 hours). PD plasma exposures (normalized to the 100 mg dose level) ranged from 345–1128 ng.hr/mL for AUC 0–12 and from 36–111 ng/mL for Cmax and were consistent with those observed in prior solid tumor studies. Immunohistochemistry of BM on Day 8 (prior to initiation of BD) in 7/8 pts demonstrated preferential and complete inhibition of CDK4/6-specific phosphorylation of Rb and Ki67 in tumor cells. Follow up BMs after 21 days, showed G1-S cell cycle progression upon PD withdrawal, confirming PDs synchronization effect. Conclusions: Targeting CDK4/6 with PD to induce prolonged G1 arrest in combination with B/D represents the first mechanism-based targeting of the cell cycle in cancer, and it appears to be effective in MM. Pts are being actively accrued to Schedule B consisting of sequential PD-B/D (12 days of PD followed by B/D as in Schedule A) to assess the safety of this novel schedule and the efficacy following cell cycle synchronization. Disclosures: Niesvizky: Celgene: Consultancy, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding, Speakers Bureau; Proteolix: Consultancy, Research Funding. Courtney:Pfizer: Employment. DuFresne:Pfizer: Employment. Wilner:Pfizer: Employment. Chen:Pfizer: Employment. Mark:Celgene: Speakers Bureau; Millenium: Speakers Bureau. Coleman:Bristol-Myers Squibb Research & Development: Consultancy.

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Synergistic Loss of IRF4 and Induction of IRF7 Sensitizes Primary Myeloma Cells to IMiD Killing by IFNβ in Prolonged Early G1 Arrest Induced by CDK4/CDK6 Inhibition

Blood ◽

10.1182/blood.v120.21.572.572 ◽

2012 ◽

Vol 120 (21) ◽

pp. 572-572

Author(s):

Xiangao Huang ◽

David Jayabalan ◽

Maurizio Di Liberto ◽

Jackson D Harvey ◽

Anna C. Schinzel ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Research Funding ◽

S Phase ◽

G1 Arrest ◽

Combination Therapies ◽

Employment Equity ◽

Myeloma Cells ◽

Sequential Combination ◽

Equity Ownership

Abstract Abstract 572 The immunomodulatory drugs (IMiD) lenalidomide (Len) and pomalidomide (Pom) are effective therapies for multiple myeloma (MM), improving both disease-free and overall survival in relapsed or refractory MM with a favorable toxicity profile. However, MM remains incurable due to the eventual development of drug resistance, and the mechanism of IMiD action is not well understood. Developing novel mechanism-based combination therapies and defining the mechanism of IMiD action are thus timely and necessary. By inducing prolonged early G1 arrest (pG1) through inhibition of CDK4/CDK6 with a highly specific, potent and reversible inhibitor, PD 0332991, we have now developed a novel sequential combination therapy that both inhibits proliferation of MM cells and sensitizes them to IMiD killing. Our rationales are as follows: 1) cell cycle dysregulation underlies unrestrained proliferation of MM cells in relapse, as in other cancers; 2) dysregulation of CDK4 or CDK6, which drives cell cycle progression through early G1, precedes the increase in proliferation in MM progression; 3) inhibition of CDK4/CDK6 by PD 0332991 arrests the cell cycle in early G1 in all Rb-positive primary bone marrow myeloma cells (BMMM)s, ex vivo and in MM patients in a phase I/II clinical trial; 4) pG1 sensitizes MM cells to killing by diverse clinically relevant agents in pG1 and in subsequent synchronous S phase entry after the release of early G1 block. Our replication kinetics data show that Len induces a dose-dependent late G1 arrest by 48 hours in MM cell lines, but apoptosis and reduction of viable cells is not evident until 72 hours, and appears independent of late G1 arrest. However, killing by Len or Pom is markedly accelerated and enhanced in pG1 induced by PD 0332991 for 24 hours (twice the time needed to induce G1 arrest in MM cells). Importantly, acceleration of early G1 arrest by PD 0332991 sensitizes BMMMs to killing by Len (16/20 cases) and by Pom (3/4 cases) despite protection by bone marrow stromal cells. Thus, IMiDs preferentially target MM cells arrested in early G1, in contrast to most cytotoxic agents, which primarily target tumor cells in S phase, thereby providing a strong rationale for improving IMiD therapy by prior induction of pG1. Whole transcriptome sequencing (WTS, RNA-Seq) and q-PCR analyses of BMMMs further revealed that correlating with Len killing, genes of the interferon (IFN) signaling pathway are coordinately and prominently induced by Len, and by Len and pG1 in synergy, but not by pG1 alone. These data provide the first direct evidence for induction of IFN by IMiD and enhancement by pG1 in BMMMs, suggesting a pivotal role for IFN in mediating IMiD killing in synergy with pG1 in MM. pG1 halts scheduled gene expression in early G1 and prevents the expression of genes programmed for other cell cycle phases, as we have demonstrated by WTS in conjunction with q-PCR and immunoblot analyses. Synergistic induction of IFN may stem from the imbalance in gene expression in pG1 and its interplay with IMiD signaling. Indeed, pG1 activates the synthesis of IRF4, an essential survival factor of MM cells, but markedly amplifies the loss of IRF4 protein induced by Len or Pom through transcriptional and posttranscriptional mechanisms. This leads to induction of IRF7, a primary and direct target of IRF4 repression, and IFNb, which is activated by IRF7. The essential roles of IRF4 and IRF7 in mediating IMiD killing and pG1 sensitization by IFNb signaling have been further confirmed by shRNA silencing in representative MM cell lines that have been characterized by WTS and shown to recapitulate pG1 sensitization of Len and Pom killing. In summary, we have developed a novel sequential combination therapy that both inhibits proliferation and enhances IMiD killing of MM cells by induction of pG1 through selective CDK4/CDK6 inhibition. This therapy combines oral compounds with excellent toxicity profiles and acts in pG1; thus, it may serve as a maintenance therapy to both control tumor expansion and prevent self-renewal. This study presents the first WTS-validated therapeutic strategy in MM, and demonstrates, for the first time, that the IRF4-IRF7-IFNb pathway mediates IMiD killing and pG1 amplifies it. Further investigation may uncover novel molecular therapeutic targets and biomarkers for genome-based patient stratification for cell cycle-based IMiD combination therapies. Disclosures: Huang: Celgene, Corp: Research Funding. Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor Lenalidomide is an Immunomodulatory drug. Mark:Millenium Inc.: Speakers Bureau; Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hussein:Celgene, Corp: Employment, Equity Ownership. Randolph:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Onyx, Millemium, Celgene. Speakers bureau: Millenium and Celgene: Consultancy, Research Funding. Chen-Kiang:Bristol-Myers Squibb: Consultancy; Pfizer, Inc.: Research Funding.

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Inhibition of CDK4/CDK6 Sensitizes Myeloma to IMiD By Reducing the MEIS2 to Cereblon Ratio That Accelerates IKZF1 and IKZF3 Degradation

Blood ◽

10.1182/blood.v126.23.500.500 ◽

2015 ◽

Vol 126 (23) ◽

pp. 500-500

Author(s):

Xiangao Huang ◽

David Jayabalan ◽

Maurizio Di Liberto ◽

Zhengming Chen ◽

Anna C Schinzel ◽

...

Keyword(s):

Cell Cycle ◽

Clinical Response ◽

Research Funding ◽

Ex Vivo ◽

Cell Cycle Control ◽

Selective Inhibition ◽

Hematologic Malignancies ◽

G1 Arrest ◽

Myeloma Cells

Abstract Lenalidomide (Len) and pomalidomide (Pom) are immunomodulatory drugs (IMiDs) effective in hematologic malignancies, in combination therapies for multiple myeloma (MM) in particular. Cereblon (CRBN), a component of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Emerging evidence suggests that IMiDs bind CRBN and block an endogenous substrate MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN and their degradation. This then leads to loss of IRF4 necessary for myeloma survival. The clinical relevance of these novel findings, however, has not been defined. To address this question, we've investigated the mechanism of IMiD action and the functional consequences in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=31) in stromal co-culture ex vivo in the context of the clinical response to Len or Pom in vivo before or after biopsy. We showed by whole transcriptome sequencing, protein analysis and functional assays that 1) BMMCs are addicted to IKZF3-IRF4 for survival; 2) Len-mediated IRF4 loss leads to de-repression of IRF7, induction of interferon (IFN) response genes and TRAIL-mediated apoptosis; and 3) the magnitude of IFN induction is tightly associated with killing of BMMCs by Len. Importantly, the IMiD sensitivity in BMMCs ex vivo correlated with the prior or subsequent clinical response to IMiD-based therapies in individual myeloma patients, suggesting that the clinical response to IMiDs in myeloma is largely intrinsic to myeloma cells. IMiDs have been reported to cause cell cycle arrest. We found that before evidence of killing, Len and Pom induced late G1 arrest by both repressing CCNA2 (encoding cyclin A) mRNA synthesis and elevating p21 and p27 proteins independent of Rb and p53. This result suggests that IMiDs preferentially kill cells in G1 arrest, and that induction of p rolonged early G1 arrest (pG1) beyond the normal G1 transit time by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991, Ibrance) may sensitize MM cells to IMiD killing, as it does to killing by other agents. Indeed, induction of pG1 by palbociclib overrides cell cycle regulation by Len, and sensitizes BMMCs to Len-mediated apoptosis by augmenting the loss of IRF4 protein and the induction of IRF7, IFNb and TRAIL. Further investigation revealed that induction of pG1 by CDK4/CDK6 inhibition sensitizes primary myeloma cells to IMiD killing by rapid acceleration of Len-mediated loss of IKZF1 and IKZF3 proteins, within one hour of IMiD addition. Loss and gain of function studies demonstrates that MEIS2 opposes pG1 sensitization to Len killing; however, MEIS2 itself is regulated by the cell cycle. Induction of pG1 reduces the ratio of MEIS2 to CRBN by both reducing the MEIS2 protein rapidly and increasing the CRBN protein at a later time in cooperation with Len. In summary, our data provide the first evidence that induction of prolonged early G1 arrest by selective inhibition of CDK4/CDK6 amplifies IMiD killing of primary myeloma cells by both repressing MEIS2 and increasing CRBN protein in cooperation with Len. This leads to a profound reduction in the ratio of MEIS2 to CRBN that accelerates the loss of IKZF1, IKZF3 and IRF4, and enhances IFN and TRAIL induction. Reducing the MEIS2/CRBN ratio thus represents a novel mechanism by which CDK4/CDK6 inhibition sensitizes myeloma to IMiDs, and a means for developing mechanism-based IMiD therapy through cell cycle control. Disclosures Huang: Celgene: Research Funding. Off Label Use: Palbociclib (PD 0332991) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Rossi:Calgene: Speakers Bureau. Pearse:Celegen: Consultancy. Mark:Calgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Niesvizky:Celgene: Consultancy, Speakers Bureau. Chen-Kiang:Celgene: Consultancy.

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