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Human neoplasias are often addicted to the cellular proteasome machinery. This has led to the development of bortezomib and carfilzomib proteasome inhibitors, approved for the treatment of multiple myeloma. Cancers, however, were found resistant to the proteasome inhibition in clinical trials, suggesting effective, cancer-specific compensatory responses. Here we employed global proteomics to determine contributions of compensatory mechanisms upon the proteasome inhibition with carfilzomib - in the cells of multiple myeloma, normal fibroblasts, and cancers of lung, colon, and pancreas. A pathway-oriented siRNA screen based on proteomics results showed that molecular chaperones, autophagy- and endocytosis-related proteins are cancer-specific vulnerabilities in combination with carfilzomib. HSP70 family chaperones HSPA1A/B were the most universal proteasome inhibition responders in the proteomes of all the studied cell types and HSPA1A/B inhibition most specifically sensitized cancer cells to carfilzomib in cell lines, patient-derived organoids and mouse xenografts. Overlap of proteomics with RNA-seq data showed that the proteasome inhibition-dependent HSPA1A/B induction in cancer cells is mainly transcription-driven and HSF1/2-dependent. Consequently, we found that a high level of HSPA1A/B mRNA is associated with a low proteasome activity in cancer patient tissues and is a risk factor in cancer patients with the low level of expression of the proteasome. Functionally, the HSPA1A/B induction does not affect a proteasome expression bounce-back upon the carfilzomib treatment, while it supports other mechanisms of the proteasome inhibition response - autophagy, unfolded protein response, and directly the 26S proteasome activity. We found that the 26S proteasome is chaperoned and protected from the inhibition with carfilzomib by HSPA1A/B assisted by DNAJB1 co-chaperone in cancer cells and using purified protein system in vitro. Thus, we define HSPA1A/B as a central player in the cellular compensatory response to the decreased proteasome activity, and the sensitive target in cancer cells with the inhibited proteasome.

Proteasome machinery is a major proteostasis control system in human cells, actively compensated upon its inhibition. To understand this compensation, we compared global protein landscapes upon the proteasome inhibition with carfilzomib, in normal fibroblasts, cells of multiple myeloma, and cancers of lung, colon, and pancreas. Molecular chaperones, autophagy, and endocytosis-related proteins are the most prominent vulnerabilities in combination with carfilzomib, while targeting of the HSP70 family chaperones HSPA1A/B most specifically sensitizes cancer cells to the proteasome inhibition. This suggests a central role of HSP70 in the suppression of the proteasome downregulation, allowing to identify pathways impinging on HSP70 upon the proteasome inhibition. HSPA1A/B indeed controls proteasome-inhibition-induced autophagy, unfolded protein response, and endocytic flux, and directly chaperones the proteasome machinery. However, it does not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, targeting of NRF1 proves effective in decreasing the viability of cancer cells with the inhibited proteasome and HSP70.