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Page 8 of 20 Ho et al. J Cancer Metastasis Treat 2019;5:70 I http://dx.doi.org/10.20517/2394-4722.2019.25

EXPLOITING AUTOPHAGY IN MM
Preclinical studies of autophagy modulators in MM
HDAC6 inhibitors
[99]
Histone deacetylases (HDACs) catalyze the removal of acetyl groups on lysine residues in target proteins .
Furthermore, HDACs also deacetylate non-histone proteins, thereby providing an additional layer of
[99]
control over protein function, stability, and protein-protein interaction . HDAC6 is a class IIb HDAC that
is mainly localized to the cytoplasm, unlike class I and IIa HDACs which shuttle between the cytoplasm
[99]
and nucleus, suggesting that HDAC6 functions mainly to regulate non-histone proteins . Indeed,
HDAC6 possess intrinsic ubiquitin-binding activity and co-localizes with the microtubule network to
transport misfolded polyubiquitinated proteins to aggresomes/autophagosomes for subsequent lysosomal
degradation [99-101] . HDAC6 therefore promotes ubiquitin-dependent or ubiquitin-independent aggresome
formation and while HDAC6 is not required for the initiation of autophagy per se, it is necessary for the
targeted delivery of the autophagy machinery to aggresomes [62,100-105] . Importantly, HDAC6 also promotes
[105]
the formation of an F-actin network essential for autophagosome-lysosome fusion . The development
of HDAC6 inhibitors therefore presents an opportunity to exploit autophagy to destabilize protein
homeostasis in MM.

Preclinical studies have shown that the HDAC6-selective inhibitors WT161 and Tubacin trigger the
accumulation of acetylated tubulin of and inhibits MM cell growth in vitro [106,107] . Additionally, combinatory
treatment using WT161 or Tubacin with the PI bortezomib not only induces synergistic cytotoxicity
but was also able to overcome bortezomib resistance [106,107] . Mechanistically, the addition of WT161 to
bortezomib resulted in further accumulation of polyubiquitinated proteins which led to a further increase
in ER stress signaling and the UPR, evidenced by the upregulation of ATF4 and the pro-apoptotic protein
[106]
CHOP . Interestingly, the ER stress sensor proteins inositol-requiring enzyme 1 (IRE1α) and PRKR-like
endoplasmic reticulum kinase (PERK) were found to be downregulated by WT161, suggesting that HDAC6
[106]
inhibition also suppresses the UPR, preventing it from functioning as an ER stress mitigator .

Another HDAC6-selective inhibitor, ACY-1215, demonstrated synergistic cytotoxicity when used in
[108]
combination with carfilzomib, an irreversible second generation PI . The addition of ACY-1215 resulted
[108]
in increased LC3-II consistent with the disruption of autophagic flux secondary to HDAC6 inhibition .
Mechanistically, the addition of ACY-1215 to carfilzomib inhibited both aggresome formation and
[108]
autophagosome-aggresome association . Consistently, a novel HDAC6 inhibitor MPT0G413 was shown
[109]
to both disrupt bortezomib-induced aggresome formation and exhibit synergism with bortezomib .
The combination of MPT0G413 and bortezomib could also overcome cell adhesion-mediated drug
[109]
resistance in the MM-BMSC co-culture setting . MPT0G413 was further found to decrease MM-BMSC
adhesion and downregulate the pro-myeloma cytokines VEGF and IL-6 in the context of the MM BM
[109]
microenvironment .
Autophagy inhibitors + DNA-damaging chemotherapy
DNA damaging agents such as melphalan are highly cytotoxic to MM cells and are a mainstay of
treatment in MM, particularly as a conditioning regimen ahead of autologous hematopoietic stem-cell
[110]
transplantation . Consistent with the aforementioned link between DDR and autophagy, a recent study
found that melphalan and doxorubicin induce cytoprotective autophagy as a pro-survival mechanism in
[111]
MM cells through the upregulation of BECLIN-1-dependent autophagosomes . knockdown of autophagy
genes (BECLIN-1 and ATG5) and pharmacologic inhibition of autophagy (via hydroxychloroquine and
3-methyladenine) significantly enhanced the cytotoxicity of melphalan and doxorubicin in vitro and
[111]
in vivo .
High mobility group protein B1 (HMGB1) is a critical regulator of autophagy that is often upregulated
in MM [112,113] . Specifically, HMGB1 binds competitively to BECLIN-1 causing BCL-2 displacement and