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[112]
autophagy induction . A study found that HMGB1 overexpression in MM was associated with mTOR
[113]
inhibition, autophagy induction, and reduced sensitivity to dexamethasone . Conversely, knockdown of
[113]
HMGB1 increased apoptosis in MM cells exposed to dexamethasone .
HMGB1 has also been recognized a marker of induction of immunogenic cell death (ICD). ICD is triggered
by exposure to certain cytotoxic agents (e.g., anthracyclines, oxaliplatin, bortezomib) [114,115] . ICD involves
the release of soluble mediators along with alterations in the cancer cell surface composition in a way that
converts dying cancer cells into therapeutic vaccines that are phagocytosed by antigen presenting cells
(APCs) for the cross-priming of CD8+ T-cells to stimulate anti-tumor specific T-cell immune responses [114,115] .
HMGB1 is a nuclear nonhistone chromatin-binding protein secreted by dying tumor cells exposed to
cytotoxic agents that binds to toll-like receptor 4 on APCs to increase the rate of antigen processing
[116]
and presentation by APCs to T cells . Interestingly, studies have shown that autophagy in tumor cells
regulates HMGB1 secretion and that inhibition of autophagy leads to intracellular sequestration of HMGB1
[117]
and the induction of caspase-mediated cell death . Macroautophagy therefore enhances antigen cross-
priming after ICD which results in the following conundrum. From a cytotoxicity point of view, it makes
sense to combine DNA-damaging agents with autophagy inhibitors. However, from an ICD perspective,
the addition of autophagy inhibitors to DNA-damaging agents may be counterproductive to antigen cross-
priming.

Autophagy modulators + PI
Bortezomib, a first-in-class PI, was approved for use in MM by the FDA in 2003 and has become a
mainstay of therapy at every disease stage ever since. Despite significant clinical efficacy in most naïve
patients, most of the patients ultimately become refractory to bortezomib therapy [118-120] . This has prompted
the development of second-generation PIs (e.g., carfilzomib and ixazomib) currently in clinical use
for relapsed or refractory MM; and next-generation PIs (e.g., marizomib and oprozomib) currently in
advanced clinical trials . Based on evidence that susceptibility to PI depends on the ability of MM cells
[121]
to remove misfolded proteins through protein quality control pathways, novel therapeutic approaches
targeting alternative pathways in protein homeostasis are also actively being studied .
[122]

Research has shown that crosstalk exists between the proteasome, UPR, and autophagy [65,123,124] . Specifically,
whenever the proteasome is overwhelmed and/or inhibited, polyubiquitinated and misfolded proteins co-
aggregate in the cytosol to form aggresomes which are then degraded through macroautophagy [101,125,126] .
Mechanistically, proteasome inhibition induces ER stress and PERK-eIF2α and IRE1-JNK activation,
leading to the induction of autophagy [127,128] . Other mechanisms of bortezomib resistance, in the context
of autophagy, include the upregulation of PROFILIN-1 which enhances autophagy through BECLIN-1
interaction and increased expression of CIC5, a chloride channel that enhances bortezomib-induced
autophagy via AKT-mTOR inhibition [129] . PIs also rapidly induce the expression of SQSMT1/p62 to
upregulate p62-dependent autophagy to compensate for proteasome insufficiency .
[64]
Along the same line, preclinical studies have reported synergistic cytotoxicity with dual blockade of the
proteasome and autophagy in MM. MG132, a PI, induced cytoprotective autophagy that can be inhibited,
by 3-methyladenine, to enhance apoptotic cell death [128] . Notably, 3-methyladenine resulted in the
[128]
accumulation of polyubiquitinated proteins and exacerbation of ER stress in cells treated with MG132 .
Autophagy inhibitors that have demonstrated synergy and enhanced MM cytotoxicity with bortezomib
in vitro include Elaiphyllin (macrolide antibiotic) and Metformin [130,131] . Elaiphyllin, in particular, had
[130]
significant cytotoxic activity even when used alone in mutant p53 MM . Metformin, on the other hand,
[131]
inhibits GRP78 which is crucial mediator of bortezomib-induced protective autophagy .

However, other conflicting studies have also shown that Metformin inhibits MM proliferation by
inducing autophagy (and cell cycle arrest) through AMPK activation and dual repression of mTORC1 and