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revealed an enrichment of mutations in many of these regulators at the time of disease relapse, including a
[36]
significant percentage of samples with mutations in the H3 lysine 36 (H3K36) trimethyltransferase SETD2 .
Further investigation of the functional consequences of SETD2 mutations in cell line and murine models of
ALL revealed that normal SETD2 activity is required for recruitment of DNA damage repair machinery to
sites of DNA breaks. In the absence of SETD2-mediated H3K36 trimethylation, DNA damage repair proteins
are no longer appropriately recruited to sites of DNA damage, leading to resistance to DNA damaging
[37]
chemotherapy agents, including cytarabine, 6-thioguanine, doxorubicin, and etoposide .

PRECLINICAL AND CLINICAL USE OF EPIGENETIC MODULATORS
DNMT and HDAC inhibitors
DNMT inhibitors have gained traction in the treatment of myelodysplastic syndrome and acute myeloid
[38]
leukemia (AML), where they have been shown to effectively reactivate epigenetically silenced genes .
Preclinical studies have demonstrated the efficacy DNMT inhibitors in the context of B-ALLs harboring
MLL rearrangements. For example, Schafer et al. demonstrated global hypermethylation in MLL-
[39]
rearranged patient samples relative to other B-ALL samples and normal controls, leading to gene silencing.
In cell line assays, treatment with the DNMT inhibitor decitabine induced cell death, concomitant with re-
[39]
expression of hypermethylated genes . Given numerous studies demonstrating the importance of aberrant
DOT1L function in the pathogenesis of MLL-rearranged ALL [33,34] and genetic studies demonstrating a
[40]
dependency on DOT1L in these leukemias , more recent attention has focused on the development and
preclinical validation of DOT1L inhibitors. In cell line models of MLL-rearranged ALL, DOT1L inhibitors
have been shown to inhibit the aberrant gene expression pattern associated with DOT1L activity and to
potently induce cell death .
[41]
In addition to the use of DNMT inhibitors alone, several preclinical and clinical studies have evaluated the
efficacy of combination therapy involving DNMT inhibitors and HDAC inhibitors as a means of dually
targeting the aberrant epigenetic landscape of ALL, an approach that has demonstrated clinical efficacy
[15]
[42]
in patients with AML . Hogan et al. demonstrated a relapse-specific pattern of methylation and gene
expression that was associated with the acquisition of chemoresistance in B-ALL. This same group went
on to demonstrate the feasibility of reversing this signature with epigenetic modulators. Specifically, they
treated relapsed B-ALL cells with a combination of decitabine and vorinostat and found that previously
silenced genes were re-expressed upon treatment. Furthermore, treating cells with these two agents resulted
in dramatic sensitization to conventional chemotherapy, suggesting that epigenetic modulators may have
[43]
clinical utility in restoring chemosensitivity at the time of disease relapse . In a small phase II trial in a
mixed pediatric and adult population, this therapeutic strategy demonstrated an overall response rate of 75%
in patients who completed therapy. The authors performed methylation profiling of samples at diagnosis
and following decitabine treatment, and noted genome-wide hypomethylation following decitabine,
[44]
with significant differences in the methylation patterns of responders vs. non-responders . Notably, this
therapeutic strategy also formed the basis for a pediatric phase II Therapeutic Advances in Childhood
Leukemia and Lymphoma (TACL) clinical trial in the context of relapsed ALL (NCT01483690), though the
trial was terminated early due to toxicity.

Additional studies have focused on the use of HDAC inhibitors alone. One compound, LBH589
(panobinostat), demonstrated early in vitro and in vivo efficacy in preclinical models of ALL. Specifically,
treatment of ALL cell lines with LBH589 resulted in increased histone acetylation, which was accompanied
by a reduction in proliferation and cell viability. Furthermore, in a xenograft model, LBH589 was sufficient
to slow disease progression as a single agent and cooperated with conventional chemotherapy to result in
[45]
a further increase in therapeutic efficacy . Other preclinical studies have demonstrated that, in ALL cell
lines and in blast cells from a patient with relapsed ALL, LBH589 induced apoptosis concomitant with
[46]
an increase in the expression of genes required for the DNA damage response . A TACL phase I clinical