Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1–expressing myeloma cells

Morales, Alejo; Kurtoğlu, Metin; Matulis, Shannon M.; Liu, Jiangxia; Siefker, David; Gutman, Delia; Kaufman, Jonathan L.; Lee, Kelvin P.; Lonial, Sagar; Boise, Lawrence H.

doi:10.1182/blood-2011-01-327197

Cited by 119 publications

(145 citation statements)

References 49 publications

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“…We observed high (albeit varied) levels of Mcl-1 and Bcl-xL and low levels of Bcl-2 protein expression across the TNBC cell line panel. Although expression of a particular anti-apoptotic protein does not necessarily indicate a cell is dependent on it for survival, 13,14 significant expression is likely a necessary precondition for the protein to be an important sensor of cell death stimuli and thus a viable target for therapeutic manipulation. Indeed, all Mcl-1, Bcl-xL or dually sensitive cell lines expressed the requisite protein(s).…”

Section: Discussionmentioning

confidence: 99%

“…High expression of a pro-survival Bcl-2 family member does not necessarily correlate with dependency on that protein to prevent apoptosis. [12][13][14] Individual pro-survival Bcl-2 family proteins preferentially inhibit a subset of pro-apoptotic family members, 15 and cancer cells require a counterbalancing antagonist for whichever proapoptotic stimuli are present. Moreover, additional regulatory mechanisms, such as limiting trafficking to the mitochondria or inducing degradation, may alter activity regardless of the absolute protein level expressed.…”

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confidence: 99%

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Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer

et al. 2015

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Breast cancer is the second-most frequently diagnosed malignancy in US women. The triple-negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate programmed cell death by upregulating anti-apoptotic proteins of the B-cell CLL/lymphoma 2 (Bcl-2) family. One family member, myeloid cell leukemia-1 (Mcl-1), is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing Mcl-1 and Bcl-2-like protein 1 isoform 1 (Bcl-xL) expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset upon Mcl-1 knockdown. In contrast, Bcl-xL knockdown only modestly reduced viability, indicating that Mcl-1 is a more important survival factor. However, dual silencing of both Mcl-1 and Bcl-xL reduced viability in most cell lines tested. These proliferation results were recapitulated by BH3 profiling experiments. Treatment with a Bcl-xL and Bcl-2 peptide had only a moderate effect on any of the TNBC cell lines, however, co-dosing an Mcl-1-selective peptide with a peptide that inhibits Bcl-xL and Bcl-2 was effective in each line tested. Similarly, the selective Bcl-xL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by Mcl-1 knockdown markedly improved its EC 50 . ABT-199, which selectively inhibits Bcl-2, did not synergize with Mcl-1 knockdown, indicating the relatively low importance of Bcl-2 in these lines. Mcl-1 sensitivity is not predicted by mRNA or protein levels of a single Bcl-2 family member, except for only a weak correlation for Bak and Bax protein expression. However Breast cancer is the second-most frequently diagnosed malignancy in US women with 230 000 new cases and 40 000 deaths in 2011. The triple-negative breast carcinoma (TNBC) subtype, which does not express the estrogen receptor (ER) and progesterone receptor (PR) and lacks overexpression of human epidermal growth factor receptor-2 (HER2), afflicts nearly 15% of all breast cancer patients and remains refractory to currently available endocrine and HER2-directed therapies.1,2 The current standard of care for TNBC is radiation and neoadjuvant cytotoxic chemotherapy, and carries a poor clinical prognosis. [3][4][5] As with most cancers, TNBC cells are under metabolic and oncogenic stress and require inhibition of the intrinsic apoptotic pathway for survival. 6 Under normal physiological conditions, this pathway is tightly regulated by both pro-and anti-apoptotic members of the B-cell CLL/lymphoma 2 (Bcl-2) family. Stressors such as DNA damage, hypoxia or oncogenic signaling, cause increased expression or translocation of proapoptotic Bcl-2 family members, such as Bim, Bad and Noxa, to the mitochondria. 7 These proteins subsequently trigger pore formation in the mitochondrial outer membrane via induced multimerization of Bak or Bax, a proc...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer

et al. 2015

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“…Plasma cells were either treated in the presence of buffy coat cells or CD138C plasma cells were isolated as previously described 42 and treated. Twenty-four hour apoptosis was determined by staining with anti-CD38 (BD Biosciences 340927), anti-CD45 (BD Biosciences 348795), and Annexin V-FITC(BioVision 1001-1000).…”

Section: Patient Samplesmentioning

confidence: 99%

“…Real-time PCR was performed using TaqMan gene expression master mix (ABI 4368814) with an ABI 9600 Fast thermocycler as described previously. 42 The following probes were used: Bim (bcl2l11) Mm00437796_m1, Bid (BID) Hs00609632_m1, NOXA (PMAIP1) Hs00560402_m1, Bmf (BMF) Hs00372937_m1, Mcl-1 (mcl1) Mm00725832_s1, Bcl-2 (bcl2) Mm00477631_m1, Bcl-x L (bcl2l1) Mm00437783_m1, and GAPDH 4352932-0912031 were purchased from Applied Biosystems.…”

Section: Real Time Pcrmentioning

confidence: 99%

Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

Ponder

Matulis

Hitosugi

et al. 2016

Cancer Biology & Therapy

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Carfilzomib (Kyprolis Ò ), a second generation proteasome inhibitor, is FDA approved for single-agent use among relapsed/refractory multiple myeloma (MM). To enhance the therapeutic efficacy of carfilzomib, we sought to combine carfilzomib with other novel agents. TG02, a multi-kinase inhibitor, targets JAK2 and CDK9. The rationale for co-treatment with carfilzomib and TG02 is that both independently target Mcl-1 and most myeloma cells are dependent on this anti-apoptotic protein for survival. We observed at least additive effects using the combination treatment in MM cell lines and patient samples. To determine how the bone marrow environment affects the efficacy of the combination we conducted co-culture experiments with Hs-5 stromal cells. We also examined the mechanism of increased apoptosis by determining the affect on expression of the Bcl-2 family of proteins. We found that carfilzomib increases NOXA mRNA expression, as expected, and TG02 treatment caused a decrease in Mcl-1 protein but not mRNA levels. Consistent with this possibility, we find silencing CDK9 does not change carfilzomib sensitivity in the same manner as addition of TG02. Since changes in Mcl-1 protein occur in the presence of a proteasome inhibitor we hypothesize that regulation of Mcl-1 translation is the most likely mechanism. Taken together our data suggest that dual inhibition of Mcl-1 via decreased expression and the induction of its antagonist NOXA by the combination of carfilzomib and TG02 is active in myeloma and warrants further testing preclinically and in clinical trials. Moreover, regulation of Mcl-1 by TG02 is more complex than initially appreciated.

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“…We found that the combination of A-1210477 and cobimetinib was synergistic as shown by a mechanism that involved disruption of the interaction of MCL-1 with the pro-apoptotic BH3-only proteins BIM and BAK by A-1210477, and by the ability of cobimetinib to suppress MCL-1 and induce BIM expression. MCL-1 is known to bind to and neutralize BIM and BAK (49), and release of BIM from MCL-1 contributes to apoptosis induction. Studies have shown that BIM induction is an important mechanism by which MEK/ERK inhibitors, including cobimetinib, can cooperatively kill tumor cells (50)(51)(52).…”

Section: Discussionmentioning

confidence: 99%

Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer.

Kawakami

Huang

Pal

et al. 2017

JCO

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Oncogenic BRAF V600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAF V600E mutant CRCs, treatment failure may be related to BRAF V600E -mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAF V600E can upregulate antiapoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAF V600E -induced MCL-1 upregulation was confirmed by ectopic BRAF V600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1 Thr163 ) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylationmimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced pro-apoptotic BIM to a greater extent than did vemurafenib in BRAF V600E cell lines. MCL-1 knockdown vs control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small molecule MCL-1 inhibitor, A-1210477 also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with pro-apoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAF V600E -mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/ stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAF V600E mutant CRCs.

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Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1–expressing myeloma cells

Cited by 119 publications

References 49 publications

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer

Myeloid cell leukemia-1 is an important apoptotic survival factor in triple-negative breast cancer

Dual inhibition of Mcl-1 by the combination of carfilzomib and TG02 in multiple myeloma

Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer.

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