MARCH5 mediates NOXA-dependent MCL1 degradation driven by kinase inhibitors and integrated stress response activation
- Hematology-Oncology Division, Department of Medicine, and Cancer Center, Beth Israel Deaconess Medical Center and Harvard Medical School, United States
- Department of Urology, Gunma University Hospital, Japan
Figures
Figure 1 with 1 supplement
EGFR inhibition decreases MCL1 via NOXA-dependent mechanism.
(A) LNCaP cells were treated with EGFR inhibitor erlotinib (10 μM) for 3 hr, followed by immunoblotting. (B) LNCaP cells were transfected with three distinct NOXA siRNA or non-target control siRNA …
Figure 1—figure supplement 1
EGFR inhibition increases proteasome-dependent MCL1 degradation.
(A) LNCaP cells were pretreated with MG115 (10 μM) and MG132 (10 μM) for 30 min, followed by treatment with erlotinib for 4 hr. (B) LNCaP cells were treated with DMSO or erlotinib for 2 hr, followed …
Figure 2 with 1 supplement
EGFR inhibition upregulates NOXA through ISR activation.
(A) LNCaP cells were treated with erlotinib (10 μM) for 3 hr, followed by immunoblotting. (B) LNCaP cells were treated with erlotinib (10 μM) at time 0 and were harvested over a time course from 30 …
Figure 2—figure supplement 1
LNCaP cells were transfected with siRNA pools targeting PERK, GCN2, the combined PERK and GCN2 pools, or a nontarget control siRNA (siNC).
At 72 hr after transfection, cells were treated for 4 hr with Erlotinib (5 mM) or vehicle, followed by immunoblotting.
Figure 3 with 1 supplement
Tyrosine kinase inhibitors decrease MCL1 via mitochondria-associated E3 ligase MARCH5.
(A) LNCaP cells transfected with pooled MARCH5 siRNAs or non-target control siRNA were treated with DMSO, erlotinib (10 μM), or dinaciclib (200 nM) for 4 hr. (B) LNCaP cells transfected with pooled …
Figure 3—figure supplement 1
MARCH5 knockdown increases MCL1 in additional PCa, breast, and lung cancer cell lines.
(A) LNCaP cells transfected with pooled HUWE1 (MULE) siRNAs or non-target control siRNA were treated with erlotinib (10 μM) for 5 hr, followed by immunoblotting. (B) LNCaP cells were pretreated with …
Figure 4 with 1 supplement
EGFR inhibition enhances MARCH5-MCL1 interaction without altering MARCH5 activity.
(A) LNCaP cells were pretreated with proteasome inhibitors MG115 (10 μM) and MG132 (10 μM) for 1 hr, followed by treatment with erlotinib (10 μM) or DMSO for 3 hr. The cell lysates were subject to …
Figure 4—figure supplement 1
MARCH5 depletion increases NOXA protein.
(A and B) LNCaP cells were transfected with MARCH5 pooled siRNAs (#1, Dharmacon), an individual siRNA (#2, Fisher), or non-target control, followed by western blot (A) or qRT-PCR (B). (*, p<0.05).
Figure 5 with 1 supplement
Mitochondria-targeted agents upregulate NOXA and induce MARCH5-dependent MCL1 degradation.
(A) LNCaP cells were treated with human mitochondrial translation inhibitor actinonin (0–100 μM) for 5 hr, and whole cell lysates were then assessed by immunoblotting. (B) LNCaP cells were treated …
Figure 5—figure supplement 1
Mitochondria-targeted agents increase caspase-independent MCL1 degradation and synergize with BCLXL/BCL2 inhibitor to induce apoptosis.
(A) LNCaP cells were pretreated with caspase inhibitor Z-DEVD-FMK (20 μM) for 1 hr, followed by treatment with CPI-613 (200 μM), gamitrinib-TPP (5 μM), or erlotinib (10 μM) and BCLXL/BCL2 inhibitor …
Figure 6 with 1 supplement
MARCH5 deletion or MCL1 amplification exists in subsets of PCa patients.
(A–C) Molecular profiles (copy number alterations and mutation) of MCL1 (A), MARCH5 (B), and HUWE1 (MULE) (C) among PCa datasets in the cBioPortal for Cancer Genomics (http://cbioportal.org). (D) …
Figure 6—figure supplement 1
MARCH5 deletion is observed in subsets of PCa patients.
(A) Progression-free survival for tumors with MARCH5 deletion (deep and shallow) in TCGA data set. (B and C) Heatmap of gene alterations for MARCH5, MCL1, NOXA (PMAIP), MULE (HUWE1), and Bim (BCL2L11…
Figure 7 with 1 supplement
MARCH5 depletion sensitizes BH3 mimetics to drive apoptosis in PCa cells.
(A and B) SgMARCH5-KO or control LNCaP cells were treated with S63845 (0–20 μM) (A) or another MCL1 inhibitor AZD5991 (0–20 μM) (B) for 12 hr. Apoptosis induction was detected with cleaved caspase 3 …
Figure 7—figure supplement 1
MARCH5 shows codependency with MCL1 in DepMap CRISPR-CAS9 essentiality screens in cancer cells.
(A) Correlation between MCL1 and MARCH5 dependency scores in cancer cells from CRISPR-CAS9 screens using GeCKO libraries. (B and C) Top five genes correlated with MCL1 dependency score (B) or genes …
Additional files
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Supplementary file 1
Key Resources Table.
- https://cdn.elifesciences.org/articles/54954/elife-54954-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/54954/elife-54954-transrepform-v2.docx
