Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6

Abstract A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

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Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Blood ◽

10.1182/blood.v114.22.601.601 ◽

2009 ◽

Vol 114 (22) ◽

pp. 601-601

Author(s):

Rodger E. Tiedemann ◽

Yuan Xiao Zhu ◽

Jessica Schmidt ◽

Hongwei Yin ◽

Quick Que ◽

...

Keyword(s):

Multiple Myeloma ◽

Epithelial Cells ◽

Tumor Cells ◽

Research Funding ◽

Kinase Inhibitors ◽

Lymphoid Tissues ◽

Myeloma Cells ◽

Stat3 Phosphorylation ◽

Human Multiple Myeloma

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

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ClC5 Decreases the Sensitivity of Multiple Myeloma Cells to Bortezomib via Promoting Prosurvival Autophagy

Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics ◽

10.3727/096504017x15049221237147 ◽

2018 ◽

Vol 26 (3) ◽

pp. 421-429 ◽

Cited By ~ 7

Author(s):

Huimin Zhang ◽

Yuhui Pang ◽

Chuanbao Ma ◽

Jianying Li ◽

Huaquan Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Small Interfering Rna ◽

Critical Role ◽

Treatment Strategies ◽

Myeloma Cell ◽

Beclin 1 ◽

Myeloma Cells ◽

Multiple Myeloma Cell ◽

Interfering Rna

Resistance to bortezomib (BZ) is the major problem that largely limits its clinical application in multiple myeloma treatment. In the current study, we investigated whether ClC5, a member of the chloride channel family, is involved in this process. The MTT assay showed that BZ treatment decreased cell viability in three multiple myeloma cell lines (ARH77, U266, and SKO-007), with IC50 values of 2.83, 4.37, and 1.91 nM, respectively. Moreover, BZ increased the conversion of LC3B-I to LC3B-II and expressions of beclin-1 and ATG5, concomitantly with a decreased p62 expression. Pharmacological inhibition of autophagy with 3-MA facilitated cell death in response to BZ treatment. Additionally, BZ increased ClC5 protein expression in ARH77, U266, and SKO-007 cells. Knockdown of ClC5 with small interfering RNA sensitized cells to BZ treatment, and upregulation of ClC5 induced chemoresistance to BZ. Furthermore, ClC5 downregulation promoted BZ-induced LC3B-I to LC3B-II conversion and beclin-1 expression, whereas overexpression of ClC5 showed the opposite results in ARH77 cells. Finally, BZ induced dephosphorylation of AKT and mTOR, which was significantly attenuated by ClC5 inhibition. However, ClC5 upregulation further enhanced AKT and mTOR dephosphorylation induced by BZ. Our study demonstrates that ClC5 induces chemoresistance of multiple myeloma cells to BZ via increasing prosurvival autophagy by inhibiting the AKT‐mTOR pathway. These data suggest that ClC5 may play a critical role in future multiple myeloma treatment strategies.

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Curcumin (Diferuloylmethane) Inhibits Constitutive and IL-6-Inducible STAT3 Phosphorylation in Human Multiple Myeloma Cells

The Journal of Immunology ◽

10.4049/jimmunol.171.7.3863 ◽

2003 ◽

Vol 171 (7) ◽

pp. 3863-3871 ◽

Cited By ~ 332

Author(s):

Alok C. Bharti ◽

Nicholas Donato ◽

Bharat B. Aggarwal

Keyword(s):

Multiple Myeloma ◽

Myeloma Cells ◽

Stat3 Phosphorylation ◽

Human Multiple Myeloma

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The Effect of Plant Extracts Kalanchoe daigremontiana and Aloe arborescens on the Metabolism of Human Multiple Myeloma Cells

Biointerface Research in Applied Chemistry ◽

10.33263/briac115.1317113186 ◽

2021 ◽

Vol 11 (5) ◽

pp. 13171-13186

Keyword(s):

Multiple Myeloma ◽

Atpase Activity ◽

Tumor Cells ◽

Plant Extracts ◽

Glutathione Level ◽

Myeloma Cells ◽

Human Multiple Myeloma ◽

Aloe Arborescens ◽

Kalanchoë Daigremontiana ◽

Kalanchoe Daigremontiana

The effect of plant extracts Kalanchoe daigremontiana, and Aloe arborescens on human multiple myeloma cells' viability was investigated. It was shown that plant extracts of kalanchoe and aloe reduced tumor cells' viability by 13 and 46%, respectively. The combination of plant extracts with doxorubicin showed an additive synergism of action, enhancing the antitumor effect. Treatment of multiple myeloma cells with plant extracts led to a decrease in intracellular reduced glutathione level. The intracellular glutathione level decreased by 25% under the action of kalanchoe extract and by 63% under the action of aloe extract. Extracts from kalanchoe and aloe decreased mitochondrial membrane potential by 19 and 53%, respectively. The results of the study showed that kalanchoe extract increased ATPase activity, but aloe extract did not affect the level of ATPase activity. The results showed that plant extracts of kalanchoe and aloe affect tumor cells' metabolism and contribute to their death. It was concluded that herbal extracts Kalanchoe daigremontiana and Aloe arborescens have antitumor activity, and aloe extract is more effective than kalanchoe.

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