Circumvention of Mcl-1-dependent drug resistance by simultaneous Chk1 and MEK1/2 inhibition in human multiple myeloma cells.

The anti-apoptotic protein Mcl-1 plays a major role in multiple myeloma (MM) cell survival as well as bortezomib- and microenvironmental forms of drug resistance in this disease. Consequently, there is a critical need for strategies capable of targeting Mcl-1-dependent drug resistance in MM. The present results indicate that a regimen combining Chk1 with MEK1/2 inhibitors effectively kills cells displaying multiple forms of drug resistance stemming from Mcl-1 up-regulation in association with direct transcriptional Mcl-1 down-regulation and indirect disabling of Mcl-1 anti-apoptotic function through Bim up-regulation and increased Bim/Mcl-1 binding. These actions release Bak from Mcl-1, accompanied by Bak/Bax activation. Analogous events were observed in both drug-naïve and acquired bortezomib-resistant MM cells displaying increased Mcl-1 but diminished Bim expression, or cells ectopically expressing Mcl-1. Moreover, concomitant Chk1 and MEK1/2 inhibition blocked Mcl-1 up-regulation induced by IL-6/IGF-1 or co-culture with stromal cells, effectively overcoming microenvironment-related drug resistance. Finally, this regimen down-regulated Mcl-1 and robustly killed primary CD138+ MM cells, but not normal hematopoietic cells. Together, these findings provide novel evidence that this targeted combination strategy could be effective in the setting of multiple forms of Mcl-1-related drug resistance in MM.

CDK inhibitors upregulate BH3-only proteins to sensitize human myeloma cells to BH3 mimetic therapies.

BH3 mimetic drugs induce cell death by antagonizing the activity of antiapoptotic Bcl-2 family proteins. Cyclin-dependent kinase (CDK) inhibitors that function as transcriptional repressors downregulate the Bcl-2 family member Mcl-1 and increase the activity of selective BH3 mimetics that fail to target this protein. In this study, we determined whether CDK inhibitors potentiate the activity of pan-BH3 mimetics directly neutralizing Mcl-1. Specifically, we evaluated interactions between the prototypical pan-CDK inhibitor flavopiridol and the pan-BH3 mimetic obatoclax in multiple myeloma (MM) cells in which Mcl-1 is critical for survival. Coadministration of flavopiridol and obatoclax synergistically triggered apoptosis in both drug-naïve and drug-resistant MM cells. Mechanistic investigations revealed that flavopiridol inhibited Mcl-1 transcription but increased transcription of Bim and its binding to Bcl-2/Bcl-xL. Obatoclax prevented Mcl-1 recovery and caused release of Bim from Bcl-2/Bcl-xL and Mcl-1, accompanied by activation of Bax/Bak. Whether administered singly or in combination with obatoclax, flavopiridol also induced upregulation of multiple BH3-only proteins, including BimEL, BimL, Noxa, and Bik/NBK. Notably, short hairpin RNA knockdown of Bim or Noxa abrogated lethality triggered by the flavopiridol/obatoclax combination in vitro and in vivo. Together, our findings show that CDK inhibition potentiates pan-BH3 mimetic activity through a cooperative mechanism involving upregulation of BH3-only proteins with coordinate downregulation of their antiapoptotic counterparts. These findings have immediate implications for the clinical trial design of BH3 mimetic-based therapies that are presently being studied intensively for the treatment of diverse hematopoietic malignancies, including lethal multiple myeloma.

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2.

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

Disruption of IkappaB kinase (IKK)-mediated RelA serine 536 phosphorylation sensitizes human multiple myeloma cells to histone deacetylase (HDAC) inhibitors.

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) ß-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/ß (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKß-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKß knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKß-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.

Bortezomib interacts synergistically with belinostat in human acute myeloid leukaemia and acute lymphoblastic leukaemia cells in association with perturbations in NF-κB and Bim.

Interactions between the histone deacetylase inhibitor belinostat and the proteasome inhibitor bortezomib were investigated in acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) cells. Co-administration of sub-micromolar concentrations of belinostat with low nanomolar concentrations of bortezomib sharply increased apoptosis in both AML and ALL cell lines and primary blasts. Synergistic interactions were associated with interruption of both canonical and non-canonical nuclear factor (NF)-κB signalling pathways, e.g. accumulation of the phosphorylated (S32/S36) form of IκBα, diminished belinostat-mediated RelA/p65 hyperacetylation (K310), and reduced processing of p100 into p52. These events were accompanied by down-regulation of NF-κB-dependent pro-survival proteins (e.g. XIAP, Bcl-xL). Moreover, belinostat/bortezomib co-exposure induced up-regulation of the BH3-only pro-death protein Bim. Significantly, shRNA knock-down of Bim substantially reduced the lethality of belinostat/bortezomib regimens. Administration of belinostat ± bortezomib also induced hyperacetylation (K40) of α-tubulin, indicating histone deacetylase inhibitor 6 inhibition. Finally, in contrast to the pronounced lethality of belinostat/bortezomib toward primary leukaemia blasts, equivalent treatment was relatively non-toxic to normal CD34(+) cells. Together, these findings indicate that belinostat and bortezomib interact synergistically in both cultured and primary AML and ALL cells, and raise the possibilities that up-regulation of Bim and interference with NF-κB pathways contribute to this phenomenon. They also suggest that combined belinostat/bortezomib regimens warrant further attention in acute leukaemias.

Disruption of Src function potentiates Chk1-inhibitor-induced apoptosis in human multiple myeloma cells in vitro and in vivo.

Ras/MEK/ERK pathway activation represents an important compensatory response of human multiple myeloma (MM) cells to checkpoint kinase 1 (Chk1) inhibitors. To investigate the functional roles of Src in this event and potential therapeutic significance, interactions between Src and Chk1 inhibitors (eg, UCN-01 or Chk1i) were examined in vitro and in vivo. The dual Src/Abl inhibitors BMS354825 and SKI-606 blocked Chk1-inhibitor-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, markedly increasing apoptosis in association with BimEL up-regulation, p34(cdc2) activation, and DNA damage in MM cell lines and primary CD138(+) MM samples. Loss-of-function Src mutants (K297R, K296R/Y528F) or shRNA knock-down of Src prevented the ERK1/2 activation induced by Chk1 inhibitors and increased apoptosis. Conversely, constitutively active Ras or mitogen-activated protein kinase/ERK kinase 1 (MEK1) significantly diminished the ability of Src inhibitors to potentiate Chk1-inhibitor lethality. Moreover, Src/Chk1-inhibitor cotreatment attenuated MM-cell production of vascular endothelial growth factor and other angiogenic factors (eg, ANG [angiogenin], TIMP1/2 [tissue inhibitor of metalloproteinases 1/2], and RANTES [regulated on activation normal T-cell expressed and secreted]), and inhibited in vitro angiogenesis. Finally, coadministration of BMS354825 and UCN-01 suppressed human MM tumor growth in a murine xenograft model, increased apoptosis, and diminished angiogenesis. These findings suggest that Src kinase is required for Chk1-inhibitor-mediated Ras → ERK1/2 signaling activation, and that disruption of this event sharply potentiates the anti-MM activity of Chk1 inhi-bitors in vitro and in vivo.

Interruption of the Ras/MEK/ERK signaling cascade enhances Chk1 inhibitor-induced DNA damage in vitro and in vivo in human multiple myeloma cells.

The role of the Ras/MEK/ERK pathway was examined in relation to DNA damage in human multiple myeloma (MM) cells exposed to Chk1 inhibitors in vitro and in vivo. Exposure of various MM cells to marginally toxic concentrations of the Chk1 inhibitors UCN-01 or Chk1i modestly induced DNA damage, accompanied by Ras and ERK1/2 activation. Interruption of these events by pharmacologic (eg, the farnesyltransferase inhibitor R115777 or the MEK1/2 inhibitor PD184352) or genetic (eg, transfection with dominant-negative Ras or MEK1 shRNA) means induced pronounced DNA damage, reflected by increased gammaH2A.X expression/foci formation and by comet assay. Increased DNA damage preceded extensive apoptosis. Notably, similar phenomena were observed in primary CD138(+) MM cells. Enforced MEK1/2 activation by B-Raf transfection prevented R115777 but not PD184352 from inactivating ERK1/2 and promoting Chk1 inhibitor-induced gammaH2A.X expression. Finally, coadministration of R115777 diminished UCN-01-mediated ERK1/2 activation and markedly potentiated gammaH2A.X expression in a MM xenograft model, associated with a striking increase in tumor cell apoptosis and growth suppression. Such findings suggest that Ras/MEK/ERK activation opposes whereas its inhibition dramatically promotes Chk1 antagonist-mediated DNA damage. Together, these findings identify a novel mechanism by which agents targeting the Ras/MEK/ERK pathway potentiate Chk1 inhibitor lethality in MM.

Interactions between bortezomib and romidepsin and belinostat in chronic lymphocytic leukemia cells.

PURPOSE: The goal of this study was to characterize interactions between the proteasome inhibitor bortezomib and the histone deacetylase (HDAC) inhibitors (HDACI) romidepsin or belinostat in chronic lymphocytic leukemia (CLL) cells. EXPERIMENTAL DESIGN: Primary and cultured (JVM-3 and MEC-2) CLL cells were exposed to agents alone or in combination, after which cell death was determined by 7-aminoactinomycin D staining/flow cytometry. Acetylation of target proteins, activation of caspase cascades, and expression of apoptosis-regulatory proteins were monitored by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) activity was determined by luciferase reporter assay. Cells were transiently transfected with wild-type and acetylation site-mutated (inactive) RelA(p65) (e.g., K221R, K310R, or K281/221/310R) and assessed for HDACI sensitivity. RESULTS: Combined exposure to very low concentrations of romidepsin or belinostat (i.e., low nanomolar and submicromolar, respectively) in combination with low nanomolar concentrations of bortezomib synergistically induced cell death in primary and cultured CLL cells. These events were likely associated with prevention of HDACI-mediated RelA acetylation and NF-kappaB activation by bortezomib, down-regulation of antiapoptotic proteins (i.e., Bcl-xL, Mcl-1, and XIAP), as well as up-regulation of the proapoptotic protein Bim, resulting in activation of caspase cascade. Finally, CLL cells transfected with inactive RelA displayed a significant increase in HDACI lethality. CONCLUSIONS: Coadministration of the clinically relevant HDACIs romidepsin or belinostat with bortezomib synergistically induces cell death in CLL cells, likely through mechanisms involving, among other factors, NF-kappaB inactivation and perturbation in the expression of proapoptotic and antiapoptotic proteins. A strategy combining HDAC with proteasome inhibition warrants further attention in CLL.

Cotreatment with suberanoylanilide hydroxamic acid and 17-allylamino 17-demethoxygeldanamycin synergistically induces apoptosis in Bcr-Abl+ Cells sensitive and resistant to STI571 (imatinib mesylate) in association with down-regulation of Bcr-Abl, abrogation of signal transducer and activator of transcription 5 activity, and Bax conformational change.

Interactions between the histone deacetylase (HDAC) inhibitors suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein (Hsp) 90 antagonist 17-allylamino 17-demethoxygeldanamycin (17-AAG) have been examined in Bcr-Abl(+) human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571 (imatinib mesylate). Cotreatment with 17-AAG and SAHA or SB synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI571, as well as in CD34(+) cells isolated from the bone marrows of three patients with chronic myelogenous leukemia. These events were associated with increased binding of Bcr-Abl, Raf-1, and Akt to Hsp70, and inactivation of extracellular signal-regulated kinase 1/2 and Akt. In addition, 17-AAG/SAHA abrogated the DNA binding and the transcriptional activities of signal transducer and activator of transcription (STAT) 5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Cotreatment with 17-AAG and SAHA also induced down-regulation of Mcl-1, Bcl-xL, and B-Raf; up-regulation of Bak; cleavage of 14-3-3 proteins; and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Moreover, ectopic expression of Bcl-2 attenuated cell death induced by this regimen, implicating mitochondrial injury in the lethality observed. Together, these findings raise the possibility that combining HDAC inhibitors with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl(+) leukemias, including those resistant to STI571.

The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells.

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-beta-D-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27(KIP1), cyclins A, E, and D(1), and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH(2)-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger L-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

Enforced expression of the tumor suppressor p53 renders human leukemia cells (U937) more sensitive to 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis.

The effects of enforced expression of p53 on the sensitivity of p53(-/-) human monocytic leukemia cells (U937) to apoptosis following exposure to the S-phase-specific antimetabolite 1-[beta-D-arabinofuranosyl]cytosine (ara-C) were examined. Cells were stably transfected with a plasmid containing a chimeric DNA construct encoding a temperature-sensitive p53 variant (135(ala-->val)), which transactivates at 32 degrees but is non-functional at 37 degrees. A significant reduction in the S-phase population was observed in ptsp53 mutants incubated at 32 degrees. Nevertheless, while vector controls did not exhibit differential sensitivity to ara-C at 32 degrees versus 37 degrees, temperature-sensitive p53 mutants displayed a significant increase in apoptosis at the permissive temperature. This was not accompanied by increased ara-CTP formation, DNA incorporation of [3H]ara-C, or altered expression of Bcl-2 or Bax. Enhanced sensitivity was associated with increased mitochondrial injury (e.g. cytochrome c release), caspase activation, and loss of clonogenic survival. Significantly, ptsp53 cells synchronized in S phase were markedly more sensitive to ara-C-mediated mitochondrial injury and apoptosis at 32 degrees, indicating that wild-type p53 specifically enhances the susceptibility of this subpopulation to ara-C lethality. Consistent with these results, transient transfection of human wild-type p53 cDNA rendered parental U937 cells more sensitive to ara-C-mediated cell death. Collectively, these findings indicate that p53 expression renders S-phase U937 cells more susceptible to ara-C-mediated mitochondrial dysfunction, cytochrome c release, apoptosis, and loss of clonogenic survival without enhancing ara-C metabolism. Such findings raise the possibility that loss of functional p53 activity allows leukemia cells to circumvent ara-C lethality.