scholarly journals CRISPR/Cas9 mediated knock-out of VPREB1 gene induces a cytotoxic effect in myeloma cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245349
Author(s):  
Mai Khaled ◽  
Amr S. Moustafa ◽  
Nashwa El-Khazragy ◽  
Maha Imam Ahmed ◽  
Marwa Ali Abd Elkhalek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cytotoxic Effect ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Knock Out ◽  
Surrogate Light Chain ◽  
Protein Levels ◽  
Mrna And Protein Expression

Background Multiple Myeloma (MM) is a heterogeneous, hematological neoplasm that accounts 2% of all cancers. Although, autologous stem cell transplantation and chemotherapy are currently the most effective therapy, it carries a notable hazards, in addition for being non curative. Recently, the Clustered Regular Interspaced Short Palindromic Repeats (CRISPR-cas9) has been successfully tried at the experimental level, for the treatment of several hematological malignancies. Objectives We aimed to investigate the in-vitro effect of CRISPR-cas9-mediated knock-out of V-set pre B-cell surrogate light chain 1”VPREB1” gene on the malignant proliferation of primary cultured myeloma cells. Methods Bioinformatics’ analysis was performed to explore the gene expression profile of MM, and the VPREB1 gene was selected as a target gene for this study. We knocked-out the VPREB1 gene in primary cultured myeloma cells using CRISPR-cas9, the VPREB1 gene editing efficacy was verified by determining VPREB1 gene expression at both the mRNA and protein levels by qPCR and immunofluorescence, respectively. Furthermore, the cytotoxic effect on primary myeloma cells proliferation was evaluated using cytotoxicity assay. Results There was a statistically significant reduction of both VPREB1 mRNA and protein expression levels (p<0.01). knock-out of VPREB1 gene in myeloma cell line resulted in a statistically significant reduction of myeloma cell proliferation. Conclusion CRISPR-cas9-mediated knock-out of VPREB1 gene is effective for inhibiting the proliferation of primary myeloma cells. This would provide a basis for a promising therapeutic strategy for patients with multiple myeloma.

Proangiogenic properties of human myeloma cells: production of angiopoietin-1 and its potential relationship to myeloma-induced angiogenesis

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 638-645 ◽  
Author(s):  
Nicola Giuliani ◽  
Simona Colla ◽  
Mirca Lazzaretti ◽  
Roberto Sala ◽  
Giovanni Roti ◽  
...  
Keyword(s):  
Myeloma Cell ◽  
Vascular Endothelial ◽  
Myeloma Cells ◽  
Vessel Formation ◽  
Protein Levels ◽  
Mrna And Protein Expression ◽  
Endothelial Growth Factor ◽  
Angiopoietin 1

AbstractPatients with multiple myeloma (MM) have increased bone marrow (BM) angiogenesis; however, the proangiogenic properties of myeloma cells and the mechanisms of MM-induced angiogenesis are not completely clarified. The angiopoietin system has been identified as critical in the regulation of vessel formation. In this study we have demonstrated that myeloma cells express several proangiogenic factors, and, in particular, we found that angiopoietin-1 (Ang-1), but not its antagonist Ang-2, was expressed by several human myeloma cell lines (HMCLs) at the mRNA and the protein levels. In a transwell coculture system, we observed that myeloma cells up-regulated the Ang-1 receptor Tie2 in human BM endothelial cells. Moreover, in an experimental model of angiogenesis, the conditioned medium of HMCLs significantly stimulated vessel formation compared with control or vascular endothelial growth factor (VEGF) treatment. The presence of anti-Tie2 blocking antibody completely blunted the proangiogenic effect of XG-6. Finally, our in vitro results were supported by the in vivo finding of Ang-1, but not Ang-2, mRNA and protein expression in purified MM cells obtained from approximately 47% of patients and by high BM angiogenesis in patients with MM positive for Ang-1, suggesting that the angiopoietin system could be involved, at least in part, in MM-induced angiogenesis.

ANKHD1 Silencing Induces S-Phase Arrest and Modulates Cell Cycle Gene Expression in Human Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1828-1828
Author(s):  
Anamika Dhyani ◽  
Adriana S S Duarte ◽  
Patricia Favaro ◽  
Sara T Olalla Saad
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Lentiviral Vector ◽  
Myeloma Cell ◽  
S Phase ◽  
Cell Cycle Gene ◽  
Myeloma Cells ◽  
Protein Levels

Abstract Abstract 1828 ANKHD1 is a multiple ankyrin repeats containing protein with a single KH domain. It is a large protein (∼ 280 kDa) derived from an 8 kb transcript. The ANKHD1 gene, present in human chromosome 5q31.3 as a single copy is ubiquitously expressed in normal human tissues and reported to be highly expressed in cancers, such as acute leukemia. Previous study showed higher expression of ANKHD1 in bone marrow plasma cells (CD138+) from Multiple Myeloma patients as compared to control (1) and it is also over expressed in multiple myeloma cell lines such as MM1S, MM1R, U266 and RPMI 8266 at both mRNA and protein level (2). However, the functional role of ANKHD1 in myeloma cells is unknown. In the present study, by silencing ANKHD1 gene expression in glucocorticoid resistant (U266) and sensitive (MM1S) myeloma cell lines, we studied its effect on cell cycle, proliferation and apoptosis. For gene silencing, specific shRNA-expressing lentiviral vector targeting the ANKHD1 gene and as negative control, sequence specific to Lac z gene were used. Cell growth was measured using the MTT colorimetric assay, whereas for apoptosis and cell cycle analysis Flow cytometry was used. Western blot and RTPCR were used for studying gene expression and protein levels, respectively. The results showed that lentiviral vector containing coding sequences for shRNA significantly downregulated ANKHD1 gene expression in Multiple Myeloma cells at the mRNA and the protein levels (p<0.05). Furthermore, we found that the cell cycle was arrested at S phase and the cell proliferation was significantly inhibited in both cell lines studied (p<0.05). However, ANKHD1 suppression did not induce apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. To address the mechanism of the antiproliferative effect of ANKHD1 silencing, we examined the effect of ANKHD1 inhibition on cell cycle-related gene expression and proteins. ANKHD1 suppression caused downregulation of CDKN1B (p27), CCNB1 (cyclin B1), CDC25, CCNE1 (cyclin E1) and WEE 1 gene expression. There was no significant change in CCNA2 (Cyclin A2), CDC20 expression at mRNA levels. On the other hand, expression of CDKN1A (p21),which inhibits cyclin dependent kinases (CDKs) and plays role in preventing proliferation, was highly upregulated in both the cell lines. At protein levels, expression of Cdk2,Cdk4, p27 (CDKN1B) and E2F1 was decreased in both the cell lines with almost complete inhibition of expression in U266 cells. Taken together, the above results suggest that accumulation of cells in S phase (S phase arrest) can be due to inhibition of CDKs which binds with cyclins and are responsible for progression of cell cycle. Further, this inhibition of CDKs could be associated to increased induction of (CDKN1A) p21 in both cell lines. In conclusion, the present study demonstrates that the suppression of ANKHD1 potently inhibits proliferation and promotes cell cycle arrest without affecting rate of apoptosis in both glucocorticoid resistant as well as sensitive multiple myeloma cells. Also, as ANKHD1 suppression prevents S to G2/M progression, ANKHD1 protein might have role in cell cycle control by modulating cell cycle gene expression in intra S phase check point. The mechanisms modulating expression of these genes are under investigation. Further studies with combination of drugs that induce apoptosis and suppression of ANKHD1 may be an effective strategy for treatment of cancers, and therefore needed to be explored. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of sphingosine kinase 2 downregulates the expression of c-Myc and Mcl-1 and induces apoptosis in multiple myeloma

Blood ◽  
2014 ◽  
Vol 124 (12) ◽  
pp. 1915-1925 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Ningfei An ◽  
Robert Stuart ◽  
Luciano J. Costa ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Specific Inhibitor ◽  
Sphingosine Kinase ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Sphingosine Kinase 2 ◽  
Key Points

Key Points SK2 is overexpressed in myeloma cells and contributes to myeloma cell survival and proliferation. SK2-specific inhibitor promotes proteasome degradation of Mcl-1 and c-Myc and inhibits myeloma growth in vitro and in vivo.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

Frequent Detection of Minimal Residual Disease (MRD) in Autografts from Patients with Multiple Myeloma during Remission Using a New Culture Method.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4877-4877
Author(s):  
Yossi Cohen ◽  
Yzhar Hardan ◽  
Arnon Nagler ◽  
Dov Zipori
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cell ◽  
Residual Disease ◽  
Culture Method ◽  
Expansion Method ◽  
Myeloma Cell ◽  
Current Treatment ◽  
Myeloma Cells ◽  
The Moment

Abstract Current treatment of multiple myeloma includes autologous stem cell transplantation. However, it is unknown at the moment what is the extent of graft contamination with clonotypic myeloma cells. In order to evaluate the extent of residual contamination of the graft with myeloma cells, we used our new myeloma cell culture and expansion method developed in the Weizmann Institute of Science for the detection of MRD. We observed readily growing residual myeloma cells in 6 of seven cases, confirmed by clonal markers (FACS, PCR and FISH). However, there was some variability in the pattern of growth; one case of plasma cell leukemia and two cases with t(4;14) showed earlier and more pronounced growth, whereas one case with systemic amyloidosis and another case with MGUS failed to grow in this culture. We are currently arranging a multicenter study for further assessment of these findings and aim to answer the question whether the culture can distinguish between multiple myeloma and other plasma cell dyscrasias. Another goal is to correlate the pattern of in vitro growth of multiple myeloma cells, with clinical and chromosomal characteristics. Figure Figure

Inhibition of HIF1A Signaling by a Novel Class of Sulfonanilides for Targeted Treatment of Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2856-2856 ◽  
Author(s):  
Dirk Hose ◽  
Anja Seckinger ◽  
Hartmut Goldschmidt ◽  
Tobias Meiβner ◽  
Blanka Rebacz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromosomal Aberrations ◽  
Myeloma Cell ◽  
Proliferation Index ◽  
The Novel ◽  
Myeloma Cells ◽  
Signaling Inhibitors ◽  
Exposure Levels

Abstract Abstract 2856 Poster Board II-832 BACKGROUND. Molecular profiling of multiple myeloma allows the identification of novel targets, including HIF1A, and evaluation of their expression within large cohorts of patients. We report here the expression of HIF1A in myeloma and for the first time the preclinical testing of 4 members of a novel class of sulfonanilide HIF1A signaling inhibitors. PATIENTS AND METHODS. Expression of HIF1A was assessed using Affymetrix DNA-microarrays in 329 samples of CD138-purified myeloma cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). Proliferation of primary myeloma cells (n=67) was determined by propidium iodine staining. The effect of the novel HIF1A signaling inhibitors ELR510490, ELR510454, ELR510444 and ELR105813 on the proliferation of 12 human myeloma cell lines and the first three on the survival of 5 primary myeloma cell-samples cultured within their microenvironment was tested, and their ability to inhibit HIF1A signaling was examined using a cell-based reporter assay. Studies were also conducted to determine in vitro stability (in plasma and microsomes), as well as single-dose PK (SDPK) parameters and maximum tolerated dose (MTD) levels after dosing in mice. RESULTS. We found (i) HIF1A to be expressed by 95.4% of CD138-purified primary myeloma cell samples from previously untreated patients. (ii) HIF1A expression shows a weak but significant correlation (r=0.3, p<0.001) with a gene expression based proliferation index. (iii) Of the chromosomal aberrations tested, myeloma cells of patients with presence of a translocation t(4,14) show a significantly higher expression of HIF1A (p<0.001) vs. patients without. Myeloma cells of hyperdiploid patients show a significantly lower expression of HIF1A (p=0.02) vs. non hyperdiploid patients. (iii) HIF1A expression does not show a correlation with event-free or overall survival. (iv) The sulfonanilides ELR510490, ELR510444, ELR510454 and ELR105813 completely inhibit proliferation of all tested myeloma cell lines at nM concentrations. (v) The compounds tested, i.e. ELR510490, ELR510444, ELR510454, are active on all primary myeloma cell-samples tested. (vi) The compounds show a pronounced effect on the HIF1A signaling pathway at EC50s of 1-25nM. (vii) Pre-clinical pharmacology data for the compounds ELR510444 and ELR510490 in mice indicate favorable absorption, distribution, metabolism, and excretion (ADME) profiles as well as exposure levels upon dosing at well-tolerated levels that are significantly above the in vitro EC50 in all the cell lines tested. CONCLUSION. HIF1A is expressed in almost all primary myeloma cells. The novel HIF1A signaling inhibitors tested are very active on myeloma cell lines as well as primary myeloma cells and show favorable in vivo profiles with exposure levels in mice significantly higher than the concentrations required for the inhibition of cell proliferation or apoptosis induction in vitro. This class of compounds thus represents a promising weapon in the therapeutic arsenal against multiple myeloma. Disclosures: Rebacz: ELARA Pharmaceuticals: Employment. Lewis:ELARA Pharmaceuticals: Employment. Schultes:ELARA Pharmaceuticals: Employment.

A gp130 Interleukin-6 Transducer-Dependent SCID Model of Human Multiple Myeloma

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4727-4737 ◽  
Author(s):  
Cosette Rebouissou ◽  
John Wijdenes ◽  
Patrick Autissier ◽  
Karin Tarte ◽  
Valerie Costes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Scid Mice ◽  
Term Survival ◽  
Myeloma Cells ◽  
Tumoral Cells ◽  
Human Myeloma Cells

Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs) were used in SCID mice to grow myeloma cells whose survival and proliferation is dependent on gp130 transducer activation. The agonist anti-gp130 MoAbs neither bound to murine gp130 nor activated murine cells and, as a consequence, did not induce interleukin-6 (IL-6)–related toxicities in mice. They have a 2-week half-life in vivo when injected in the peritoneum. The agonist antibodies made possible the in vivo growth of exogenous IL-6–dependent human myeloma cells as well as that of freshly explanted myeloma cells from 1 patient with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks after myeloma cell graft and weighed 3 to 5 g. They grew as solid tumors in the peritoneal cavity and metastasized to the different peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral cells were detected in blood and bone marrow of mice grafted with the XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the phenotypic characteristics of the original tumoral cells and their in vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs. Myeloma cells from 4 patients with medullary involvement persisted for more than 1 year as judged by detectable circulating human Ig. However, no tumors were detected, suggesting a long-term survival of human myeloma cells without major proliferation. These observations paralleled those made in in vitro cultures as well as the tumor growth pattern in these patients. This gp130 transducer-dependent SCID model of multiple myeloma should be useful to study various therapeutical approaches in multiple myeloma in vivo.

scholarly journals Fate of Ikaros in Multiple Myeloma Cells upon Treatment with Lenalidomide and Proteasome Inhibitor

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3281-3281
Author(s):  
Saravanan Ganesan ◽  
Nithya Balasundaram ◽  
Hamenth Kumar Palani ◽  
Ansu Abu Alex ◽  
Sachin David ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Proteasome Inhibition ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Ubiquitinated Proteins ◽  
Two Agents ◽  
Autophagy Pathway

Abstract Recent evidences suggests that the efficacy of Lenalidomide (LEN) depends upon its ability to degrade IKZF1 and IKZF3 proteins via cereblon dependent ubiquitin proteasome pathway [Science. 2014 Jan 17; 343(6168): 301-305]. Based on this model it would theoretically be antagonistic to combine LEN with proteasome inhibitors (PI). However, it is well recognized that there is significant synergism when LEN is combined with PI and this combination is routinely and effectively used in the clinic. The mechanism of synergy and the fate of IKZF1 and IKZF3 when these two agents are combined is poorly understood. We undertook a series of experiments to study the fate of IKZF1 when this combination of drugs was used in multiple myeloma cells. Combining LEN (1uM) along with bortezomib (BTZ; 1nM) a PI showed a significant kill on U266 cells (myeloma cell line) on day 5 post treatment (n=3; P=0.02) when compared to either of the agents alone. In an MTT assay, the synergism was well documented with a combination index of 0.5 (Fig 1a). Next we assessed the function of proteasome (chymotrypsin activity) when LEN was combined with PI. We observed that LEN alone does not interfere with proteasome activity. It was noted that BTZ alone at the concentration used (5 nM) was able to effectively inhibit the activity of proteasome (Fig 1b). It was also observed that combining these two agents does not interfere with BTZ action in inhibiting proteasome complex (Fig 1b). As a result of efficient proteasome inhibition, we observed an accumulation of ubiquitinated proteins in the BTZ and LEN + BTZ treated cells when compared to control and LEN alone treated cells (Fig 1c). Next, we looked for the fate of IKZF1 in U266 cells treated with LEN, BTZ and in combination of both the drugs. As reported, we observed a degradation of IKZF1 in U266 cells upon treatment with LEN. While we did not see any degradation of IKZF1 in BTZ alone treated cells. It was noted that in combination treated cells (LEN+BTZ) there was a degradation of IKZF1 (Fig 1c). In spite of significant proteasome complex inhibition, degradation of IKZF1 was observed which suggested a proteasome independent mechanism. It is well known that proteasome inhibition results in upregulation of the autophagy pathway which in turn can degrade the accumulated ubiquitinated proteins. We noted that upon treatment with BTZ or LEN+BTZ an induction of autophagy was observed, as evidenced by an increase in generation of LC3II bands on an immunoblot (Fig 1c). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pre-treated the U266 cells with an autophagy inhibitor (3-methyladenine) followed by treatment with LEN and BTZ and noted an accumulation of IKZF1 proteins (Fig 1d). We also observed a downregulation of IKZF1 target genes IRF4 and c-MYC by 12 and 24 hours in the combination treated cells (data not shown). Taken together this data demonstrates that there is (i) significant in-vitro synergism between the two agents (ii) the combination additively induces autophagy pathway (iii) IKZF1 protein can be degraded via this autophagy pathway in the presence of effective proteasome inhibition. While additional mechanisms of synergy between these two agents cannot be excluded, further enhancing autophagy pathway in these cells by drugs like sirolimus (autophagy inducer in myeloma cells) could potentially improve the synergy between these two drugs. Disclosures No relevant conflicts of interest to declare.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

AT-101, a Small Molecule Inhibitor of Bcl-2 Family Proteins, Has Significant In Vitro Activity in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1581-1581
Author(s):  
Shaji Kumar ◽  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Clinical Trials ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Time Dependent ◽  
Myeloma Cells ◽  
Molecule Inhibitor ◽  
Apoptotic Proteins

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. Studies have shown elevated expression of anti-apoptotic proteins of the Bcl-2 family in MM cells, which appear to correlate with resistance to therapy with certain drugs. Hence, accelerating the apoptotic process by targeting the Bcl-2 family of proteins appears to be an attractive strategy for the treatment of MM. AT-101 is an orally bioavailable derivative of gossypol in cancer clinical trials, and is being developed by Ascenta Therapeutics. AT-101 behaves as a small molecule inhibitor of Bcl-2 and Bcl-XL, binding to the BH3-binding pocket of these proteins and inhibiting their ability to suppress the activity of pro-apoptotic proteins, resulting in apoptosis. Methods and Results: AT-101 was cytotoxic to several different myeloma cell lines with a median effect observed at around 5μM concentration using an MTT cell proliferation assay. Additionally, at similar doses AT-101 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. AT-101 was cytotoxic to myeloma cells under these conditions as well with a median effect at concentrations of 5–10μM. AT-101 was able to induce apoptosis in myeloma cells in a dose- and time dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. AT-101 also resulted in cytotoxicity of freshly isolated primary patient myeloma cells, inducing apoptosis in a dose dependent manner. We also studied the effect of AT-101 on levels of different pro- and anti-apoptotic proteins using flow cytometry on permeabilized cells. A time-dependent increase in the level of BAX was observed following treatment with AT-101 without any associated change in levels of Bcl-xL or Bcl-2. Further studies evaluating the combination of AT101 with other active myeloma agents as well as a detailed evaluation of its mechanisms in myeloma are ongoing. Conclusion: AT-101 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. More importantly, it has activity against cell lines resistant to conventional anti-myeloma agents. In addition, Phase I studies with this agent are currently ongoing in patients with solid tumors. The results from these studies form the rationale for early phase clinical trials in MM, either alone or in combination with other active therapies.

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