Bone Marrow Myeloma Cells Research Articles

Selective Inhibition of CDK4 and CDK6 Primes Chemoresistant Myeloma Cell for Cytotoxic Killing through Induction of Cell Cycle-Coupled Mitochondrial Dysfunction and Apoptosis

Dysregulation of cyclin-dependent kinase (CDK)4 or CDK6 activity by gain of function or loss of inhibition is one of the most frequent aberrations in cancer. This includes multiple myeloma (MM), where overexpression of CDK4 (CDK6) precedes unrestrained proliferation of CD138+ bone marrow myeloma cells in vivo, in particular during aggressive tumor growth and relapse. In complex with the D-type cyclin, CDK4 and CDK6 promote cell cycle entry and progression through G1 by inactivating the retinoblastoma protein Rb and antagonizing the INK4 family of CDK inhibitors, suggesting that inhibition of CDK4/6 is a promising approach for cell cycle control in MM. We have now developed a novel approach to both halt cell proliferation and enhance cytotoxic killing of MM cells by selective inhibition of CDK4/6 in combination with cytotoxic agents. We show that knocking down CDK4 and CDK6 expression by shRNA interference or inhibiting CDK4/6 activity with PD 0332991, the only known CDK4/6-specific small molecule inhibitor, leads to sustained G1 arrest and induction of synchronous cell cycle progression upon removal of PD 0332991. Induction of sustained early G1 arrest is not accompanied by apoptosis. However, it primes MM cells for synergistic killing by low dose cytotoxic agents of diverse modes of action, which is further augmented during synchronous S phase entry. Most importantly, induction of sustained G1 arrest with PD 0332991 primes freshly isolated chemoresistant CD138+ bone marrow myeloma cells for killing by low dose proteasome inhibitors in the presence of bone marrow stromal cells. Synergistic killing by PD 0332991 combined with low dose bortezomib (2–6 nM) in early G1 (referred to as PD-B) is mediated by increased neutralization of Mcl-1 and Bcl-2 in the absence of Noxa, as PD-B augments bortezomib activation of Bim and Mcl-1 transcription while silencing Noxa in early G1. This leads to aggregation of Bak, but not Bax, on the mitochondria, mitochondrial membrane depolarization, preferential release of Smac/DIABLO, but not cytochrome c, from mitochondria, reduction of c-IAP and caspase-9 activation. Apoptosis is further amplified through activation of caspase-8 without inducing TRAIL, FASL and TNF-α, the major ligands that trigger the extrinsic apoptosis pathway. Cytotoxic killing by PD-B is recapitulated in synergistic tumor suppression in animal models. Collectively, our ex vivo and in vivo data demonstrate that PD-B induces synergistic killing of MM cells through cell cycle-coupled regulation of Bcl-2 family genes and induction of mitochondrial dysfunction. As PD 0332991 is orally bio-available, potent and low in toxicity, our approaches have formed the basis for an ongoing, first-inclass Phase I/II clinical trial to selectively target CDK4/6 with PD 0332991 in combination with bortezomib and dexamethasone in multiple myeloma. Selective targeting CDK4 and CDK6 in combination with cytotoxic killing, therefore, provides a new and promising mechanism-based therapeutic strategy for multiple myeloma and potentially other cancers.

Silencing Immunoglobulin Gene Enhancers as a Potential Treatment Strategy for Multiple Myeloma

Background: Primary translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 occur in 60–75% of myeloma cases. This results in oncogenes from translocated chromosomes being overexpressed by linkage to the strong IgH enhancers. Specifically inhibiting the activity of a single translocated oncogene product (e.g. FGFR3 in t(4;14)+ myeloma) has been shown to decrease proliferation and increase apoptosis only in myeloma cells harboring the relevant translocation. Several recurrent translocations have been identified in myeloma, and in many cases the partner loci are non-recurrent or unidentified. No single translocation partner has been found in more than 15–20% of myelomas, and more than one overexpressed gene on the partner locus can be pathogenic (e.g. FGFR3 and MMSET). This limits the widespread applicability and potential effectiveness of therapies that directly target only one IgH-translocated oncogene. We hypothesize that silencing the IgH enhancers is a potential treatment strategy for all IgH translocation-positive myelomas, regardless of the oncogenes involved in the translocation. We further hypothesize that by specifically targeting the B/plasma cell specific transcription factors Oct-2 and Bob-1 that partially drive the IgH enhancer, a therapy could be designed with minimal toxicity to normal tissues apart from B-lineage cells.Methods: Expression of Oct-2 and Bob-1 genes and proteins were assayed with qRT-PCR and Western blot in 6 myeloma cell lines harbouring different (or no) IgH translocations, in AutoMACS-purified CD138+ and CD138− bone marrow cells from 36 patients, and in bone marrow core biopsies from 30 patients with immunohistochemistry. Using RNA interference (RNAi), we inhibited the expression of Bob-1 in KMS-11, a myeloma cell line that overexpresses fibroblast growth factor receptor 3 (FGFR3) due to a t(4;14) translocation. The effects of Bob-1 RNAi on FGFR3 expression, cell viability and induction of apoptosis were assayed by MTS and Annexin V/propidium iodide flow cytometry assays respectively.Results: Oct-2 and Bob-1 are ubiquitously expressed in myeloma cell lines and patient plasma cells, irrespective of translocation status. Both mRNA and protein expression are much greater in CD138+ myeloma bone marrow cells than in CD138− cells, and vary among patients. We will present updated results on a larger patient cohort at the meeting, along with clinical correlations including the prognostic significance of Oct-2 and Bob-1 expression levels.In keeping with decreased activity of the IgH enhancer, a decrease in the level of FGFR3 protein was observed following Bob-1 RNAi knockdown in KMS-11. Furthermore, Bob-1 knockdown led to a reduction in cell viability, and an increase in apoptosis.Discussion: Overall, these results demonstrate the potential of Bob-1 and possibly Oct-2 as therapeutic targets for the treatment of translocation-positive myeloma, due to both their contribution to overexpression of translocated oncogenes, as well as their selective and high expression in the plasma cell population. Clinically applicable approaches to Bob-1 or Oct-2 inhibition are under consideration in our laboratory.

Silencing Immunoglobulin Gene Enhancers as a Potential Treatment Strategy for Multiple Myeloma

Background: Primary translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 occur in 60–75% of myeloma cases. This results in oncogenes from translocated chromosomes being overexpressed by linkage to the strong IgH enhancers. Specifically inhibiting the activity of a single translocated oncogene product (e.g. FGFR3 in t(4;14)+ myeloma) has been shown to decrease proliferation and increase apoptosis only in myeloma cells harboring the relevant translocation. Several recurrent translocations have been identified in myeloma, and in many cases the partner loci are non-recurrent or unidentified. No single translocation partner has been found in more than 15–20% of myelomas, and more than one overexpressed gene on the partner locus can be pathogenic (e.g. FGFR3 and MMSET). This limits the widespread applicability and potential effectiveness of therapies that directly target only one IgH-translocated oncogene. We hypothesize that silencing the IgH enhancers is a potential treatment strategy for all IgH translocation-positive myelomas, regardless of the oncogenes involved in the translocation. We further hypothesize that by specifically targeting the B/plasma cell specific transcription factors Oct-2 and Bob-1 that partially drive the IgH enhancer, a therapy could be designed with minimal toxicity to normal tissues apart from B-lineage cells. Methods: Expression of Oct-2 and Bob-1 genes and proteins were assayed with qRT-PCR and Western blot in 6 myeloma cell lines harbouring different (or no) IgH translocations, in AutoMACS-purified CD138+ and CD138− bone marrow cells from 36 patients, and in bone marrow core biopsies from 30 patients with immunohistochemistry. Using RNA interference (RNAi), we inhibited the expression of Bob-1 in KMS-11, a myeloma cell line that overexpresses fibroblast growth factor receptor 3 (FGFR3) due to a t(4;14) translocation. The effects of Bob-1 RNAi on FGFR3 expression, cell viability and induction of apoptosis were assayed by MTS and Annexin V/propidium iodide flow cytometry assays respectively. Results: Oct-2 and Bob-1 are ubiquitously expressed in myeloma cell lines and patient plasma cells, irrespective of translocation status. Both mRNA and protein expression are much greater in CD138+ myeloma bone marrow cells than in CD138− cells, and vary among patients. We will present updated results on a larger patient cohort at the meeting, along with clinical correlations including the prognostic significance of Oct-2 and Bob-1 expression levels. In keeping with decreased activity of the IgH enhancer, a decrease in the level of FGFR3 protein was observed following Bob-1 RNAi knockdown in KMS-11. Furthermore, Bob-1 knockdown led to a reduction in cell viability, and an increase in apoptosis. Discussion: Overall, these results demonstrate the potential of Bob-1 and possibly Oct-2 as therapeutic targets for the treatment of translocation-positive myeloma, due to both their contribution to overexpression of translocated oncogenes, as well as their selective and high expression in the plasma cell population. Clinically applicable approaches to Bob-1 or Oct-2 inhibition are under consideration in our laboratory.

Survival Signals, Growth Cytokines, Immunosuppressive Factors and Their Cellular Perpetrators in the Myeloma Tumor Microenvironment.

Multiple myeloma (MM) is an incurable neoplasia of terminally differentiated plasma cells in the bone marrow. Essential interactions of MM cells with host bone marrow stromal cells (BMSC) induce growth factors essential for MM progression and pathogenesis, as well as induce an immunosuppressive environment that inhibits endogenous and therapeutically-induced immune responses against the MM cells. However, despite their importance, little is known about the identity of these BMSC cells or the molecular basis of their interaction with myeloma cells. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma disease progression. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with cellular partners that express the B7 (CD80/CD86) ligands. Potential candidates would include CD86+ myeloma cells themselves or B7+ dendritic cells (DC) that are known to be closely associated with myeloma cells in the patient bone marrow. When myeloma-myeloma interactions were disrupted by using the high affinity CD80/CD86 blocker CTLA4Ig (Abatacept®), increased sensitivity to arsenic trioxide (ATO) and melphalan (MEL) was observed in all the three MM cell lines U266, RPMI8226 and MM1S. For U266 viability was 93% in media alone, 84% with CTLA4Ig (100 μg/ml) alone, 86% with 2 μM ATO alone and was significantly reduced to 36% with CTLA4Ig + ATO. Similar drops in viability were observed with 25 μM MEL in combination with CTLA4Ig (33% as opposed to 71–74 % with CTLA4Ig or MEL alone). Our data suggests that this does not involve the downregulation of anti-apoptotic proteins Bcl-2, Bcl-xL or Mcl-1, commonly associated with drug resistance in myeloma. In the second part of the study, we demonstrate that myeloma cell lines or primary CD138+ myeloma cells can enhance via direct contact the ability of human monocyte derived immature DC to produce the immunosuppressive tryptophan depleting enzyme indoleamine 2,3 dioxygenase (IDO, as estimated by kynurenine (Kyn) (a tryptophan catabolite) levels in the supernatant) and also the pro-plasma cell survival cytokine IL-6. In co-cultures of IFNg treated immature DCs with either MM cell lines or with primary CD138+ myeloma cells from patient BM aspirates, the activity of IDO was enhanced ~ 2–8 fold (81 mM kyn with U266 and 20–43mM with primary cells) over that observed in control IFNg-treated DCs (9.7 mM Kyn). Western analysis also demonstrated increased IDO expression relative to IFNg activated DC controls. Blocking MM-CD28 with (Fab)2 fragments of anti-hCD28 mAb 9.3 downregulated IDO activity (9.3 mM) close to that of control, demonstrating the involvement of MM-CD28 in these interactions. We also demonstrated a significant up-regulation of the pro-myeloma survival cytokine IL-6 when immature DCs were co-cultured with CD28+ MM1S (90–300 pg/ml), a 4–9 fold increase over that of DC only control (25 – 35 pg/ml). This was further enhanced when immature DCs cultured with IL-10 (+ GM-CSF + IL-4) was used in co-cultures with MM-1S (800 – 1300 pg/ml), or with primary CD138+ myeloma cells from patient bone marrow aspirates (128–1142 pg/ml). In conclusion, our data demonstrates that blocking myeloma-CD28 - myeloma-CD86 “autocrine” interaction can enhance drug cytotoxicity, while interactions with DCs produce the essential growth cytokines IL-6 and immunosuppressive enzyme IDO with potential implications in MM survival and immune escape. Use of clinically approved agents (e.g. Abatacept®) to block myeloma-CD28 binding to its B7 ligands (increase chemotherapeutic efficacy), 1-MT to inhibit IDO and targeting DCs in the microenvironment to disrupt the tumor microenvironment could be viable therapeutic strategies for the future treatment of multiple myeloma.

Primary structure of a monoclonal κ chain in myeloma with light chain deposition disease

Previous data suggest that structural abnormalities of immunoglobulin light chains may be responsible for non-amyloid light chain deposition disease (LCDD). We report on the complete primary sequence deduced from complementary (c)DNA analysis of a normal-sized kappa chain in a case of myeloma-associated LCDD. The patient's urine contained a kappa type Bence-Jones protein made of monomers and dimers of an unglycosylated kappa chain. The bone marrow myeloma cells contained intracellular kappa and gamma chains by immunofluorescence. Biosynthesis experiments showed the production of normal-sized gamma chains and of kappa chains with the same apparent molecular mass (Mr) in SDS gels as the urinary kappa chain (26,000-27,000). These kappa chains were secreted as assembled IgG molecules and as a large excess of free monomers and dimers. The complete sequence of two identical cDNA clones derived from a normal-sized kappa messenger RNA indicated that this kappa chain belonged to the rare V kappa IV subgroup. The kappa mRNA had an overall normal structure made up of the V kappa IV sequence rearranged to J kappa 1 and followed by a normal constant exon of the Km(3) allotype. The variable region differed from the V kappa IV-J kappa 1 germline sequence by 17 amino acid substitutions. The peculiar sequence of the variable region of this kappa chain of a rare subgroup might relate to its tissue deposition.

Targeting Cdk4/6 in combination therapy of chemoresistant multiple myeloma

8503 Background: Deregulation of Cdk4 or Cdk6 is a hallmark in cancer. However, conventional cancer therapy centers on empirical cytotoxic killing. Targeting the cell cycle with broad-spectrum inhibitors has proven ineffective so far due to cross- reactivity with cell survival and the transcriptional machinery. In myeloma, over-expression of Cdk4-cyclin D1 or Cdk6-cyclin D2 predisposes primary bone marrow (BM) myeloma cells to proliferation in vivo. Conversely, silencing Cdk4/6 by its physiologic inhibitor, p18INK4c, is required for cell cycle termination during the generation of normal plasma cells. Targeting Cdk4/6, therefore, represents a rational strategy to control disease progression and drug resistance in myeloma. Methods: An orally bioactive, small molecule PD 0332991 selectively and reversibly inhibits Cdk4/6 (IC50 ∼ 60 nM) in primary myeloma cells ex vivo. This leads to exclusive G1 cell cycle arrest unaccompanied by apoptosis by BrdU labeling and apoptotic assays. Results: By inducing synchronous S phase entry upon release of G1 block or prolonging G1 arrest, inhibition of Cdk4/6 by PD 0332991 profoundly sensitizes primary myeloma cells to killing by proteasome inhibitors bortezomib and NPI-0052, or dexamethasone. Sequential treatments with PD 0332991 and bortezomib rapidly reduces tumor burden in animal models. Importantly, it overcomes chemoresistance in myeloma cells isolated from refractory relapse patients, despite stromal protection. Synergistic killing of myeloma cells lies in induction of cell cycle-specific mitochondria depolarization. Base On this basis and a favorable outcome of Phase I clinical trial, a Phase I/II PD 0332991-bortezomib clinical trial for myeloma is in progress. Conclusion: Targeting Cdk4/6 by PD 0332991 in combination with a selective cytotoxic agent represents a new class of promising cell cycle-based therapy in myeloma, and possibly other cancers. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Ortho Biotech Amgen, Merck, Millennium, Novartis Johnson & Johnson MMRF, NIH/NIAMS, Ortho Biotech, VA Merit Review

Stromal Cell-Derived Factor-1 Promotes Myeloma Cell Growth in Both Autocrine and Paracrine Manners

������������������������������������������������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������ Background: The question as to whether stromal cell-derived factor-1 (SDF-1) stimulated myeloma cell growth has been controversial. We explored the possibility that SDF-1 may function as an autocrine growth factor of myeloma cells. Methods: CD138+ primary bone marrow myeloma cells and myeloma cell lines (RPMI8226, U266, and ARH77) were used. Chemotaxis in response to SDF-1 of the cells was analyzed using TranswellsTM . Cell proliferation was measured by a colorimetric assay. SDF-1 mRNA expression was analyzed by RT-PCR (reverse-transcription-polymerase chain reaction). SDF-1 and interleukin-6 (IL-6) receptor expression as well as signaling molecule phosphorylation levels, were examined using Western blot analysis. Concentrations of SDF-1 in the cell culture supernatants were measured by ELISA assay. Results: SDF-1 alone had no discernible effect on the proliferation of CD138+ primary myeloma cells or myeloma cell lines. In contrast, SDF-1 significantly enhanced IL-6-induced proliferation of these cells. SDF-1 up-regulated the expression of IL-6 receptor and enhanced phosphorylation of AKT in an additive manner with IL-6. Co-culture of the myeloma cells with umbilical vein endothelial cells over-expressing the SDF-1 gene revealed that SDF-1 played an important role in not only the migration of the cells underneath the stromal cells but also the proliferation of the cells in contact with stromal cells. All the myeloma cell lines expressed SDF-1 mRNA, and SDF-1 was detected in the culture supernatants of the cells. The G protein-coupled receptor inhibitor, pertussis toxin, inhibited the proliferation of these cells in suspension cultures. Conclusion: SDF-1, most likely in concert with IL-6, enhanced the proliferation of myeloma cells in a both paracrine and autocrine manner. (Korean J Hematol 2008;43:127-137.)

An Analysis of Occurrence and Prognosis Related Factors of Renal Dysfunction in Patients with Multiple Myeloma.

Objective To analysis the occurrence and prognosis related factors in renal dysfunction with multiple myeloma(MM).Methods Seventy-four cases with MM were enrolled in this study. The risk factors of occurrence and prognosis were analyzed.Results The incidence of renal dysfunction (RD) with MM was 56.8%, Age, hypertention, hemoglobin, serum ALB and GLO levels, serum β2MG, serum calcium and phosphonium level, the percentage of myeloma cells in bone marrow, types of MM, Durie-Salmon stage were the single factors associated with the incidence of RD with MM. Hypertention, serum β2MG and ALB levels were the multiple factors associated with the incidence of RD with MM. ALB was the protection factor and the other two were risk factors. The renal function recovered rapidly in the patients who received CR or received blood transfusion. The patients with renal dysfunction survived shorter (28±5months) than those with normal renal function (42±6months). Renal dysfunction caused more MM patients death(84.6%) in 3 months.Conclusion Hypertention and high tumor burden were the risk factors of renal dysfunction in MM, effective chemothemapy and support treatment help renal function recovery.

AlphaVBeta3 (αvβ3) Integrin Drives the Osteoclastogenesis through a Osteoclast-Like Functional Differentiation of Myeloma Cells.

Accelerated osteoclastogenesis is the major event promoting the skeletal impairment in multiple myeloma (MM). Osteoclasts (OC) are directly activated by myeloma cells (MC), although these cells themselves may apparently undergo to OC-like morphologic transformation and produce bone erosion in vitro. Since OCs exert their function and promote osteoclastogenesis through activation of several adhesion molecules, including avb3, we investigated the role of this integrin expressed by MCs in their OC-like activity in vitro. Bone marrow MCs were purified from eight patients with severe skeletal disease (group A) and from two patients without bone lesions (group B). U266 and RPMI-8226 MC lines were the controls. Semi-nested PCR assessed the CDR3 immunoglobulin (Ig) gene rearrangement, whereas OC markers including TRAcP, cathepsin-k, calcitonin-receptor, carbonic anhydrase and vATPase were evaluated by RT-PCR. The cytoskeletal rearrangement of F-actin was analyzed by immunofluorescence. av and b3 expression on MCs was evaluated by flow-cytometry, whereas bone erosion on calcium phosphate discs and number of pits was measured by dedicated software. The effect of avb3 stimulation on the activation of osteoclastogenic function was investigated by exploring the phosphorilation of transcriptional kinases and downstream molecules, as ERK1/2 and cFos, respectively. The primary role of avb3 in OC-like functional transdifferentiation was explored in MCs by siRNA silencing for both chains. Ontogenetic derivation from the B-cell lineage was confirmed by the monoclonal CDR3 rearrangement, CD138/CD38 and Pax-5 expression. Cells from patients of group A expressed OC markers, in contrast with those of group B or U266 and RPMI-8226. Formation of the F-actin ring confirmed the differentiation of MCs toward the OC-like phenotype. Cells from group A expressed av and b3 (80±7% and 75±9%) similarly to U266 and RPMI-8226 (>90% in both instances), whereas a minimal expression was demonstrated in group B (av:6±2%; b3:8±3%). avb3+ cells produced a high number of erosive pits, at variance from avb3− cells (35±8 vs. 4±1 pits/cm2 ). The highest phosphorilation of ERK1/2 and expression of cFos was revealed in patients of group A as compared to B (840±110 OD and 905±210 OD vs. 270±35 OD and 315±80 OD, p<0.0001 in both instances). Finally, the silencing of av and/or b3 chain inhibited the erosion by avb3+ cells, resulting in a reduced number of pits (7±2 pits/cm2) similar to the pattern obtained with avb3− cells. Similarly, an inhibition of both ERK1/2 and cFos was demonstrated in silenced cells from group A with values similar to those of group B. Since avb3 drives both adhesion of Ocs to extracellular matrix and activation of osteoclastogenesis, it is conceivable that avb3+ MCs may functionally transdifferentiate to OCs in response to integrin activation induced by the contact with stromal cells within the marrow environment, thus resulting in a typical OC-like behaviour. In addition, our data suggest that MCs induce this effect by avb3, since its silencing reduces the OC-like activity in vitro.

Targeting Cdk4/6 in Combination Therapy Overcomes Proteasome Inhibitor Resistance in Multiple Myeloma through Synergistic Mitochondria Depolarization.

Deregulation of Cdk4 or Cdk6 is central to the loss of cell cycle control in cancer. In myeloma, over-expression of Cdk4-cyclin D1 or Cdk6-cyclin D2 predisposes primary bone marrow (BM) myeloma cells to proliferation in vivo1. Conversely, silencing Cdk4/6 by its physiologic inhibitor, p18INK4c, is required for G1 cell cycle arrest during the generation of normal plasma cells2. Targeting Cdk4/6, therefore, is critical for the control of disease progression and drug resistance in myeloma, but no Cdk4/6-specific inhibitor was available. We have now targeted Cdk4/6 in combination therapy in myeloma using PD 0332991 (Pfizer), a novel orally bioactive, small molecule Cdk4/6-specific inhibitor. We demonstrate that as a single agent, PD 0332991 rapidly inhibits Cdk4/6 (IC50 ∼ 60 nM) and induces exclusive G1 cell cycle arrest in primary human myeloma cells in the presence of BM stromal cells. Non-invasive whole body imaging reveals that PD 0332991 nearly completely prevents tumor growth in a rapidly disseminated xenograft human myeloma model without overt toxicity3. Because PD 0332991 is reversible and does not induce apoptosis when used as a specific Cdk4/6 inhibitor, we have further developed two strategies to target Cdk4/6 in combination therapy. In the first, the release of PD 0332991-induced G1 arrest leads to synchronous S phase reentry and preferential killing of cycling myeloma cells by cytotoxic agents. In the second, prolonging PD 0332991-induced G1 arrest markedly sensitizes primary myeloma cells to killing by cytotoxic agents including the proteasome inhibitors bortezomib and NPI-0052, as well as the steroid dexamethasone. The Cdk4/6-based combination therapy rapidly reduces tumor load in animal models. More importantly, it overcomes drug resistance in primary myeloma cells isolated from refractory relapse patients despite the protection from BM stromal cells. Synergistic killing of myeloma cells lies in the synergistic induction of mitochondria depolarization. Based on this study and the favorable outcome of a PD 0332991 Phase I clinical trial, a Phase I/II PD 0332991-bortezomib clinical trial for myeloma is underway. Targeting Cdk4/6 by PD 0332991 in combination with a selective cytotoxic agent, therefore, represents the first promising cell cycle-based therapy in myeloma, and possibly other cancers.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection.Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE).Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

A Novel Orally Active Small Molecule Potently Induces G1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cdk4/6.

Cell cycle deregulation is central to the initiation and fatality of multiple myeloma, the second most common hematopoietic cancer, although impaired apoptosis plays a critical role in the accumulation of myeloma cells in the bone marrow (BM). Inhibition of Cdk4 and Cdk6 by the Cdk inhibitor, p18(INK4c), is required for the generation of normal, functional plasma cells1. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of Cdk4-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of BM myeloma cells in vivo2. These data identify Cdk4 and Cdk6 as key determinants in the loss of cell cycle control in myeloma and suggest that Cdk4/6 may be effective targets for therapeutic intervention. Here we show that by specific inhibition of Cdk4/6, the orally active small molecule PD 0332991 potently induces G1 arrest in primary BM myeloma cells ex vivo, and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transformation, and acts in concert with the physiologic Cdk4/6 inhibitor p18(INK4c). Inhibition of Cdk4/6 by PD 0332991 is not accompanied by induction of apoptosis. However, when used in combination with a second agent such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6 in multiple myeloma and possibly other B cell cancers.

A Novel Orally Active Small Molecule Potently Induces G1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cyclin-Dependent Kinase 4/6

Cell cycle deregulation is central to the initiation and fatality of multiple myeloma, the second most common hematopoietic cancer, although impaired apoptosis plays a critical role in the accumulation of myeloma cells in the bone marrow. The mechanism for intermittent, unrestrained proliferation of myeloma cells is unknown, but mutually exclusive activation of cyclin-dependent kinase 4 (Cdk4)-cyclin D1 or Cdk6-cyclin D2 precedes proliferation of bone marrow myeloma cells in vivo. Here, we show that by specific inhibition of Cdk4/6, the orally active small-molecule PD 0332991 potently induces G(1) arrest in primary bone marrow myeloma cells ex vivo and prevents tumor growth in disseminated human myeloma xenografts. PD 0332991 inhibits Cdk4/6 proportional to the cycling status of the cells independent of cellular transformation and acts in concert with the physiologic Cdk4/6 inhibitor p18(INK4c). Inhibition of Cdk4/6 by PD 0332991 is not accompanied by induction of apoptosis. However, when used in combination with a second agent, such as dexamethasone, PD 0332991 markedly enhances the killing of myeloma cells by dexamethasone. PD 0332991, therefore, represents the first promising and specific inhibitor for therapeutic targeting of Cdk4/6 in multiple myeloma and possibly other B-cell cancers.

Rituximab treatment provides no clinical benefit in patients with pretreated advanced multiple myeloma

In the present phase II study, we tested the efficacy of a single course of rituximab (375 mg/m2 on days 1, 8, 15 and 22) as treatment for relapsed myeloma. The rationale for this study was the identification of a population of clonotypic CD20+ B cells that are believed to be precursors of malignant plasma cells. In addition, CD20 was expressed on 10% and 50% of bone marrow plasma cells in two of the ten patients enrolled. Following rituximab treatment, none of the patients achieved an objective response. Two patients had stable disease at month 6, the predefined end of the study, while, at that time, two patients were classified as having progressive disease. One patient opted to withdraw from the study at month 3, at which time he had stable disease. The other five patients had to be withdrawn early from the post-treatment observation because of need of salvage therapy for progressive disease. WHO grade ≤2 toxicity was seen in four patients. Peripheral B cells significantly decreased at 3 months, while no significant change of bone marrow myeloma cells was noted at that time. Mean paraprotein levels increased slightly during follow-up but IgM levels dropped in all patients, indicating an effective targeting of normal, short-lived plasma cells. Taken together, rituximab treatment yielded significant reductions in circulating B cells and serum IgM levels but had no beneficial clinical effect.

Identification of Target Antigens Recognized in MGUS for Immunotherapeutic Approaches.

Monoclonal gammopathy of undetermined significance (MGUS) is a benign condition with 30% of patient developing symptomatic multiple myeloma (MM). The events leading to the transformation from MGUS to MM remain unclear, however several evidences have supported the idea that the immune system, in patients with MGUS, may play a role in controlling the progression to MM; and the identification of antigenic targets could open the way for future immunotherapeutic approaches to delay or prevent such progression. To identify target antigens that are recognized by the immune system in MGUS, we have screened a cDNA expression library, produced from purified CD138+ bone marrow myeloma cells, with high dilution (1:500) serum from 3 MGUS patients with stable disease for 1 to 4 years. Using this approach we were able to identify a panel of 11 gene products to which an antibody response was observed in serum from individuals with MGUS. Responses appeared to be directed against intracellular proteins involved in variety of cellular functions such as apoptosis (SON, Hip1), DNA and RNA binding (KIAA0530, GPATC4), signal transduction regulators (AKAP11), transcriptional co-repressors (IRF2BP2), developmental proteins (OFD1) and proteins involved in the ubiquitin-proteosome pathway (PSMC1). Interestingly PSMC1, the 26S subunit of proteosome, has recently emerged as a potential target for antimyeloma therapies and AKAP11 located on the 13q14.11, a locus frequently involved in the pathogenesis of aggressive MM. Importantly, two of these identified genes with antibody response have been previously identified by SEREX screenings; KIAA0530 has been reported to induce a strong antibody response in solid tumors such as head and neck, testicular, breast and colorectal cancers and SON, a protein involved in apoptosis with similarities to oncoproteins such as myc and mos, has been previously reported by us to induce an antibody response associated with a complete response after donor lymphocyte infusion in patients with MM. Our studies are now focused on testing these proteins in a series of MGUS serum at different stages of disease as well as in early stage myeloma. Although preliminary, these data open the possibility to identify target antigens that are important in the disease process of MGUS and may allow us to design future vaccines and immunotherapeutic approaches targeting these antigens in MGUS as well as in MM.

Valproic Acid Inihibts Histone Deacetylases and Proliferation and Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

Multiple myeloma remains an incurable disease in the majority of the patients and novel treatment strategies are urgently needed. A new class of drugs, the histone deacetylase (HDAC) inhibitors take influence in epigenetic modifications and have antiproliferative effects in some malignancies. Valproic acid (VPA) is an anticonvulsant drug and was recently shown to inhibit HDACs and suppress tumor growth. The drug is currently being evaluated in clinical studies in acute myeloid leukemia. Its effects on myeloma cells are unknown. The aim of this study was to evaluate the effects of VPA on proliferation, apoptosis and HDAC inhibition in multiple myeloma cell lines as well as in sorted human bone marrow multiple myeloma cells. Myeloma cell lines, OPM-2, NCI-H929, LP-1, and freshly isolated multiple myeloma cells from bone marrow aspirates were exposed to different concentrations of VPA for 4 to 72 hours. Cell proliferation, cell cycle distribution and apoptosis were assayed in reaction to the treatment. Proliferation decreased noticeably and apoptosis was induced in a dose-dependent manner in multiple myeloma cell lines as well as in freshly sorted primary myeloma cells. After 48 hours of incubation with VPA at 1 mM, approximately 46%, 52% and 25% of OPM-2, NCI-H929 and LP-1 cell lines had undergone specific apoptosis, respectively. Freshly sorted primary bone marrow myeloma cells from patients showed also specific apoptosis. In cell cycle analysis by flow cytometry, the population of cells in the G0/G1 phase increased, whereas cells in the S phase decreased in a time and dose dependent manner. Incubation of the cell line OPM-2, for example, with 1 mM VPA for 48 hours decreased the proportion of cells in the S phase from 39 % to 6 % of the total cell count and increased cells in the G0/G1 phase from 49 % to 85 %. Acetylation of histones and expression of cyclin D1 and the cell cycle regulators p21 and p27 were studied by western blot. Histone acetylation and p21 concentrations increased after VPA treatment whereas levels of p27 remained constant. A decrease in cyclin D1 concentrations was observed. Subapoptotic doses of VPA significantly decreased the production of VEGF in OPM-2 cell line. These data show that treatment with valproic acid effectively inhibits histone deacetylase activity, leading to the accumulation of acetylated histones in multiple myeloma cells. Parallel upregulation of cell cycle inhibitors like p21WAF1 was observed, together with a reduction of cyclin D1 levels. Myeloma cell proliferation was inhibited in a time and dose dependent manner and cell cycle arrest in the G0/G1 phase was induced by VPA treatment. VPA potently induced apoptosis in all human myeloma cell lines as well as in sorted primary multiple myeloma cells in a dose and time dependent manner. These results show for the first time that VPA acts as an HDAC inhibitor in multiple myeloma cells, induces G1 cell cycle arrest, potently inhibits tumor growth and markedly induces apoptosis. In addition to its direct antitumor effect, valproic acid may exert an antiangiogenic effect by reducing VEGF production in myeloma cells. These data provide the framework for clinical studies with valproic acid in multiple myeloma.

The absence of CD56 on malignant plasma cells in the cerebrospinal fluid is the hallmark of multiple myeloma involving central nervous system

By immunohistochemistry, the CD56-positive myeloma cells were detected in three (38%) bone marrow (BM) and one (13%) cerebrospinal fluid (CSF) samples from eight patients with multiple myeloma involving the central nervous system (CNS MM). Of the three patients with CD56-positive BM myeloma cells, two had CSF myeloma cells negative for CD56. In a control cohort of 84 MM patients without CNS involvement, the BM myeloma cells were CD56-positive in 68 (80%) cases (P < 0.0001). The prevalence of CD56-negative myeloma cells in the BM and CSF of our patients suggests that CD56 downregulation may play a role in the pathogenesis of CNS MM.

HLA class I, NKG2D, and natural cytotoxicity receptors regulate multiple myeloma cell recognition by natural killer cells

AbstractThe role of natural killer (NK) cells in multiple myeloma is not fully understood. Here, NK susceptibility of myeloma cells derived from distinct disease stages was evaluated in relation to major histocompatibility complex (MHC) class I, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), and UL16 binding protein (ULBP) expression. MHC class I molecules were hardly detectable on bone marrow cells of early-stage myeloma, while late-stage pleural effusion-derived cell lines showed a strong MHC class I expression. Conversely, a high MICA level was found on bone marrow myeloma cells, while it was low or not measurable on pleural effusion myeloma cells. The reciprocal surface expression of these molecules on bone marrow- and pleural effusion-derived cell was confirmed at mRNA levels. While bone marrow-derived myeloma cells were readily recognized by NK cells, pleural effusion-derived lines were resistant. NK protection of pleural effusion cells was MHC class I dependent. Receptor blocking experiments demonstrated that natural cytotoxicity receptor (NCR) and NK receptor member D of the lectinlike receptor family (NKG2D) were the key NK activating receptors for bone marrow-derived myeloma cell recognition. In ex vivo experiments patient's autologous fresh NK cells recognized bone marrow-derived myeloma cells. Our data support the hypothesis that NK cell cytotoxicity could sculpture myeloma and represents an important immune effector mechanism in controlling its intramedullary stages. (Blood. 2005;105:251-258)

Induction of Apoptosis in Multiple Myeloma by Ligands of Peroxisome Proliferator-Activated Receptor γ (PPAR-γ).

Peroxisome proliferator-activated receptor γ (PPAR-g) is a member of a nuclear receptor superfamily, which is expressed in different tumor tissues. Activation of PPAR-γ by its ligands has been shown to reduce tumor growth, interfere with tumor cell differentiation, and induce apoptosis in a variety of human malignancies including solid tumors like colon, breast, lung, liver, prostate cancer, as well as hematological malignancies like myeloid leukemia. Recently, it has been shown that both human B-lymphocytes and B-lymphomas express PPAR-γ and induce apoptosis. 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2) is a natural activator of PPAR-γ. Thiazolidinediones, including troglitazone, rosiglitazone (RGZ), and pioglitazone (PGZ), comprise a group of synthetic PPAR-γ agonists that are currently in use for the treatment of type 2 diabetes mellitus, and have revealed anti-tumor activity in vitro. We investigated in five human multiple myeloma cell lines (LP-1, U-266, RPMI-8226, OPM-2 and IM-9) and sorted human bone marrow myeloma cells whether treatment with PGZ, RGZ or 15d-PGJ2 inhibited tumor cell growth. Expression of PPAR-γ protein was demonstrated by western blot analysis in these cell lines. All 5 cell lines were sensitive to the PPAR-γ agonists. MTT assays revealed growth arrest induced by the natural activator of PPAR-γ 15d-PGJ2 and a lower antiproliferative effect with PGZ and RGZ in a dose dependent manner. At a dose of 50 μM 15d-PGJ2 cell proliferation was reduced to values between 0% and 26% in all multiple myeloma cell lines tested. In most cell lines the anti-proliferative effect was already detectable at 10 μM. At a dose level of 50 μM PGZ cell proliferation was reduced in MTT assay after 48 hours of incubation to 48% in LP-1, 52% in IM-9, 56% in OPM-2, 72% in U-266 and 77% in RPMI-8226. Comparable results were obtained with RGZ. Induction of apoptosis was indicated by annexin V staining. Cell lines were incubated with 50 μM of PPAR-γ agonists, a concentration which had been proven to be effective for growth inhibition in MTT assay before. Again, 15-dPGJ2 was more effective than PGZ and RGZ. All of the 15d-PGJ2 treated cell lines revealed specific apoptosis ranging between 60% and 92%. Apoptosis induced by PGZ in U-266, RPMI-8226-S, IM-9, and OPM-2 cell lines ranged between 17% and 43%, for RGZ it ranged between 20% and 50%. Furthermore, in sorted bone marrow plasma cells from myeloma patients induction of apoptosis was detected. Bone marrow multiple myeloma cells from five different patients were tested. The specific apoptosis rate induced by 15-dPGJ2 lay between 29% and 96%. Apoptosis induced by PGZ showed interindividual differences. In the myeloma cells from four patients the rate of specific apoptosis ranged between 9% and 28%, but in one patient induction of apoptosis was observed neither with PGZ nor with RGZ. For RGZ, the rate of apoptosis induced in the myeloma cells from the other four patients ranged between 7% and 26%. The rate of specific apoptosis induced by 15D-PGJ2 was not statistically different for sorted human bone marrow myeloma cells sensitive versus refractory to conventional chemotherapy with anthracyclines and alkylating agents (p = 0.8). This is one of the first studies evaluating PPAR-γ expression and its therapeutical implications in human multiple myeloma cells. Thiazolidinediones comprise anti-myeloma activity and should be explored further for the treatment of multiple myeloma.

A Phase II Trial of Gleevec™ in Patients with Refractory/Relapsed Myeloma.

Background: Though STI-571 (Gleevec™) was designed to specifically inhibit the bcr-abl gene product, it inhibits other receptor tyrosine kinases at physiologically attainable concentrations, including c-kit (steel factor receptor, stem cell factor receptor, SCF-R or CD 117). Studies have shown that about one-third of patients with multiple myeloma or monoclonal gammopathy of undetermined significance have plasma cells that display reactivity for c-kit. Others have shown that several myeloma cell lines and fresh myeloma bone marrow cells proliferate in response to stem cell factor. mRNA transcripts for c-kit ligand and, more commonly, its receptor have been detected in myeloma cell lines RPMI 8226, JJN3, U266 B1, NCI-H929, ARH77 and HS-Sultan by RT-PCR.Methods: Patients were eligible for study if they presented with relapsed or refractory myeloma, an ECOG performance score < 3, ability to sign informed consent, serum creatinine <3.5 mg/dL, direct bilirubin <2 mg/dL, alkaline phosphatase <750 U/L, absolute neutrophil count > .5 x 10(9)/L and platelet count > 50 x 10(9)/L. Patients were treated with Gleevec™ 400 mg po qd. The purpose of the study was to assess the response rate of the drug Gleevec™ in patients with relapsed multiple myeloma. Secondary objectives included assessment of the tolerability of the regimen in this cohort of patients and to correlation between disease response and c-kit positivity (CD117) by flow cytometry.Results: Overall, 23 patients were enrolled between 12/01 and 11/02, of which 16 were male and 12 had a prior PBSCT. Median age was 63 years (range: 49 – 82), and the baseline performance scores were 0 (8 patients), 1 (10), and 2 (4). Median time from MM diagnosis to enrollment was 57 months (range: 11 – 133). Patients were high risk with a median PCLI of 1.3 (range: 0 – 23%) and a median beta-2 microglobulin of 4.6 (range: 1.4 – 10.1). Eleven of 21 tested patients had positive CD117 (≥20%) staining of their bone marrow plasma cells by flow cytometry. None were PDGF positive. The treatment was well tolerated. After treatment with Gleevec™, there were only 3 non-hematologic grade 3 or 4 toxicities, and they occurred in 1 patient: he developed a capillary leak type syndrome, ascites, and dyspnea as part of his terminal phase of myeloma which included evolution into plasma cell leukemia. Grade 3 myelosuppression was observed in a minority of patients: thrombocytopenia (3), neutropenia (1), and anemia (3). There were no responses, and no patients are currently receiving treatment. Patients ended treatment due to progressive disease (18 patients), death on study (3), adverse reactions (1), and other reasons (1). The median duration of treatment was 48 days (range: 12 to 349). Of the 7 patients who remained on study for more than 60 days, CD117 status was as follows: positive (4); negative (3); not ascertained (1). As of August 2004, 12 patients have died.Conclusions: Though forty-eight percent of the enrolled myeloma patients were CD117 positive, providing a putative target for the Gleevec™, no responses were seen in this high risk population of relapsed patients. Further study will be necessary to determine the significance of the observed stabilization of disease in 36% of the CD117 positive patients.