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for incorporation into DNA. Notably, CAD has been found to be upregulated in both cell lines and clinical
MDS samples that are resistant to HMAs, although this has not been a universal finding [35,98,99] .

A number of additional determinants of response to HMAs have also been identified. Mutations in genes
regulating methylation, including TET2, IDH1/2, and DNMT3A, have been associated with improved
responses to HMAs in myeloid malignancies [100-104] , although this effect has not been universally observed.
The variations in conclusions among studies have been attributed to an interaction between different
mutations and disease heterogeneity, as suggested by the relationship between lower clonal burden of
secondary mutations and higher response rate [105] .

As evidence of discordance between DNA methylation status and clinical response accumulated, the effects
of HMAs on RNA were also explored [106] . About 90% of azacitidine is incorporated into RNA, raising
the possibility that alterations in RNA metabolism might contribute to azacitidine action [107] . Consistent
with this hypothesis, azacitidine response has been linked to NSUN1, an RNA:5-methylcytosine (m5C)
methyltransferase that binds BRD4 and RNA-polymerase-II to form chromatin structures that are not
sensitive to azacitidine [108] . Bone marrow specimens from patients with azacitidine-resistant AML or MDS
showed a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin as compared
to specimens from patients with sensitive AML or MDS [108] . This resistance mechanism is not applicable to
decitabine, which contains 5-aza-cytosine bound to 2’-deoxyribose.

VENETOCLAX
BCL2 family members and regulation of apoptosis
Apoptosis is a regulated cell death process in eukaryotes that is activated by either extrinsic or intrinsic
stimuli. Apoptosis mediates well-conserved decomposition of cellular macrostructures through activation
of caspases, cysteine proteinases that cleave next to aspartate residues [109,110] . Within the intrinsic pathway
of apoptosis, cell death is initiated by mitochondrial outer membrane permeabilization (MOMP) that
facilitates the release of cytochrome c, formation of cytosolic apoptosome complexes, and subsequent
caspase activation. The MOMP process is regulated by BCL2 family members, which dynamically
modulate pro- and anti-apoptotic signaling [110,111] . Among the pro-apoptotic signaling proteins are BAK
and BAX, which permeabilize the mitochondrial outer membrane upon activation [110-112] . These are in turn
neutralized when they are bound and sequestered by the anti-apoptotic paralogs BCL2, BCLX , MCL1,
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BCLW, BCLB, and BFL1 [110-113] . A third group of BCL2 family members called BH3-only proteins includes
BID, BAD, BIK, BIM, PUMA, HRK, NOXA, and BMF, which act through competitive inhibition of the
anti-apoptotic BCL2 family members, direct activation of BAK and BAX, or a combination of these two
processes [110-114] . Upregulation of anti-apoptotic signaling (e.g., through overexpression of BCL2 and its kin)
has been implicated in the pathogenesis of cancer, particularly in hematopoietic malignancies, where the
upregulation serves as both an essential hallmark of lymphoma- or leukemogenesis and a mechanism of
drug resistance [115,116] . BCL2 is, therefore, an important therapeutic target for pharmacological inhibition in
leukemia and other malignancies [117-120] .

Action of single-agent venetoclax in hematological malignancies
BH3 mimetics are small molecules that have been developed to overcome apoptosis resistance in various
neoplasms. These agents were designed to specifically inhibit members of the anti-apoptotic pathway by
mimicking the BH3 domain found in BH3-only proteins. Like these pro-apoptotic proteins, BH3-mimetics
bind the BH3 binding groove of anti-apoptotic proteins, thereby inhibiting ability of anti-apoptotic family
member to bind and neutralize BAX and/or BAK.

The first BH3 mimetic to enter clinical trials was navitoclax (ABT-263), which was designed to
specifically mimic BAD binding to BCL2, BCLX , and BCLW [121] . Navitoclax showed efficacy in chronic
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