Resistance to HSP90 inhibition involving loss of MCL1 addiction

Abstract

Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

Figure 1

Requirement for functional mitochondria for ganetespib-induced apoptosis. (a) WT and DKO^BAX/BAK cells were treated with ganetespib 200 nm for 48 h. PARP cleavage was measured by western blot. (b) Viability was assessed by MTT assay after 72 h. WT: IC₅₀=67.77 nm; DKO^BAX/BAK IC₅₀>500 nm. (c) BAX was transiently overexpressed in DKO^BAX/BAK cells and 24 h post-transfection cells were treated with ganetespib 200 nm for a further 48 h. BAX expression and PARP cleavage were analysed by western blot. (d) MSTO-211H cells were transfected with siNT, siBAX, siBAK and the combination of siBAX and siBAK. Twenty-four hours following transfection, cells were treated with ganetespib for a further 48 h. (e) H460 stably expressing shRNA targeting BAX and BAX was treated with ganetespib 200 nm for 48 h and PARP cleavage analysed. (f) MSTO-211H, H460 and H23 have been transfected with siRNAs targeting BH3-only protein. Twenty-four hours after transfection cells have been treated with ganetespib 200 nm for further 48 h and Caspase 3 activity measured. Data were normalized to siNT control (MSTO-211H: siBID ***P=0.0001 siBIK ***P=0.0007; siPUMA **P=0.0023; H460: siBID ***P=0.0006 siBIK *P=0.0163; H23: siBID *P=0.0123 siBIK *P=0.0242; siPUMA *P=0.0481).

Figure 2

MCL1 is transcriptionally regulated by ganetespib. (a) MSTO-211H, H460 and H23 have been treated with ganetespib 200 nm for 48 h. PARP cleavage and the expression of the pro-survival BCL-2 family members have been assessed by western blot. (b) MCL1 mRNA expression was evaluated by qRT-PCR on RNA extracted from MSTO-211H, H460 and H23 cells treated for 48 h with ganetespib 200 nm. Data were normalized to untreated control (MSTO-211H: **P=0.0033; H460 *P= 0.0150; H23: ***P=0.0006). (c) The MCL1 promoter activity was measured by a luciferase reporter assay in MSTO-211H, H460 and H23 cells transfected with pGL2 basic (EV) or pGL2-MCL1 (FL) and then treated with ganetespib 200 nm for 24 h. Data were normalized to untreated full-length (FL) control (MSTO-211H: *P=0.0119; H460 ****P<0.0001; H23: ***P=0.0010). (d) MSTO-211H cells were transfected with three fragments of the promoter and treated with ganetespib 200 nm for 24 h. The MCL1 promoter activity was measured by a reporter assay. Data were normalized to untreated FL control (Fragment A: **P=0.0031; Fragment B **P=0.0077; Fragment C: ****P<0.0001). (e) The effect of siSTAT5A and sip53 on MCL1 mRNA expression and MCL1 promoter activity was measured by qRT-PCR on RNA extracted from MSTO-211H and NCI-H28 cells transfected for 24 h or luciferase reporter assay. Cells treated with ganetespib 200 nm were used as a positive control. Data were normalized to untreated control (qRT-PCR: MSTO-211H, siSTAT5A: *P=0.03977, sip53: n.s.; NCI-H28, siSTAT5A: *P=0.0439, sip53: n.s. Luciferase assay: MSTO-211H, siSTAT5A: ***P<0.0001, sip53: n.s.; NCI-H28, siSTAT5A: ***P=0.0003, sip53: n.s.). (f) The effect of ganetespib on STAT5 was measured by western blot.

Figure 3

MCL1 suppression and MCL1 addiction correlate with sensitivity to HSP90 inhibition. (a) IC₅₀ values for ganetespib in 16 cell lines (9 mesothelioma and 7 lung adenocarcinoma) have been correlated to the ability of the drug to downregulate MCL1, **P=0.0039. Number of cell lines downregulating MCL1 n=11, Number of cell lines not downregulating MCL1 n=5. (b) Explants derived form two mesothelioma patients were treated with ganetespib 2 μm for 24 h. Tissues have been stained with caspase 3 and MCL1. In patient #1, ganetespib induced apoptosis and downregulation of MCL1 (caspase 3 *P=0.0382; MCL1 **P=0.0069) and in patient #2, no significant apoptosis or MCL1 downregulation was observed. (c) MSTO-211H, H460 and H23 have been transfected with siMCL1 20 nm for 48 h. PARP cleavage and the expression of the pro-survival BCL-2 family members have been assessed by western blot. (d) MCL1 addiction has been tested in 16 cell lines and then correlated to the IC₅₀ values for ganetespib; **P=0.0095. Number of cell lines addicted to MCL1 n=8 and Number of cell lines not addicted to MCL1 n=8.

Figure 4

MCL1 dependence is lost in cells selected for resistance to ganetespib. (a) Cells selected for resistance (STAR) were tested for cell viability after 72 h treatment with ganetespib compared with parental MSTO-211H. (b) Parental and resistant cells were treated for 48 h with ganetespib 200 nm. On-target activity of ganetespib on Akt and ERK and the effect of HSP90 inhibition on pro- and anti-apoptotic proteins were analysed by western blot. (c) Cytochrome-c release was assessed after 48 h treatment with ganetespib 200 nm in the presence or absence of digitonin. Mitochondrial-free cytosolic fraction has been used for western blot analysis. (d) Resistant cells were transfected with siRNA targeting MCL1 for 48 h. Induction of apoptosis was measured by western blot with PARP antibody.

Figure 5

The combination of ganetespib and ABT737 overcomes acquired resistance through exploitation of MCL1 downregulation. (a) STAR cells were treated with ganetespib 200 nm, ABT737 200 nm or a combination of both for 48 h. PARP cleavage and MCL1 expression were measured by western blot. The effect on colony formation was measured by clonogenic assay. STAR cells were treated for 24 h with ganetespib 200 nm, ABT737 200 nm or a combination of both. After being washed, colonies were let to grow for 12 days, then fixed in methanol and stained with crystal violet. (b) Mice bearing established STAR tumours (n=7 per group) were dosed with vehicle, 100 mg/kg ABT263 (5 × /week), 100 mg/kg ganetespib (1 × /week), or the combination of ABT263 and ganetespib. Tumour volumes were assessed at end of the study. The combination of ABT263 with ganetespib resulted in a statistically significant decrease in tumour volume compared with vehicle control (error bars ±s.e.m.). (2-way Anova repeated measurements results: 19 days, ABT 263 + Ganetespib vs Vehicle, **P=0.0095; 24 days, ABT 263 + Ganetespib vs Vehicle, **P=0.0017; 27 days, ABT 263 + Ganetespib vs Vehicle, ****P<0.0001, ABT 263 + Ganetespib vs Ganetespib P=0.0132, ABT 263 + Ganetespib vs ABT 263 P=0.0004). The average body weight loss at end of the study was −7.1% with vehicle, −9.1% with ganetespib, −12.1% with ABT263 and −9.6% for the combination. (c) STAR cells were treated with ganetespib 200 nm, ABT199 200 nm, or a combination of both for 48 h. A combination of ganetespib and ABT737 in STAR and ABT199 in DOHH2 were used as a positive control. PARP cleavage and MCL-1 expression were measured by western blot. (d) STAR cells have been transfected with siRNAs targeting BH3-only protein. Twenty-four hours after transfection, cells have been treated with ganetespib 200 nm and ABT737 200 nm for further 48 h and caspase 3 activity measured. Data were normalized to siNT control (*P=0.0146). PARP cleavage, BAX, BAK, BID and PUMA expression was assessed by western blot. (e) STAR cells were transfected with siRNA targeting MCL1. Twenty-four hours after transfection, cells were left untreated or treated with ABT737 both for further 48 h. PARP cleavage and MCL1 expression were measured by western blot. (f) NCI-H28 cells were treated with ganetespib 200 nm, ABT737 200 nm or a combination of both for 48 h. PARP cleavage and MCL1 expression were measured by western blot. (g) NCI-H28 cells were transfected with siRNA targeting MCL1. Twenty-four hours after transfection, cells were left untreated or treated with either ABT737 or ganetespib for further 48 h. PARP cleavage and MCL1 expression were measured by western blot.

Figure 6

Schematic representation of the HSP90 inhibition-induced apoptosis in sensitive cells and in the context of acquired resistance. In sensitive cells, HSP90 inhibition targets the BH3-only proteins BID, BIK and PUMA and the pro-survival BCL-2 family protein MCL1 (white boxes). HSP90 inhibition requires complex interplay of these proteins to mediate BAX/BAK-dependent apoptosis. In resistant cells, MCL1 repression is conserved, however, BIK is not essential. To achieve apoptosis both the activation of BID and PUMA by HSP90 inhibition, and the targeting of the pro-survival proteins BCL-xL and BCL-w by ABT737, are needed.