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Page 4 of 11 Zhang et al. J Cancer Metastasis Treat 2020;6:21 I http://dx.doi.org/10.20517/2394-4722.2020.40

Figure 3. Graphical summary of the systems workflow used to dissect the mechanism of action for the mTOR inhibitor (mTORi)
and HDAC inhibitor (HDACi) drug combination [19] . Initial ANOVA analysis from our gene expression profiling data started with 1647
differentially expressed genes. Weighted gene co-expression network analyses determined that there were 901 genes in the entire drug
response network. Of these 901 genes, 126 genes could be assigned to the drug combination network. These genes were then evaluated
for enrichment in myeloma vs. normal samples from the same patient (GEO databases) and by multivariate prediction modeling to
assess their association with patient survival. 37 disease-specific genes were chosen for further analyses. When the data for the 37 genes
(PatentUS2014357660-A1) was evaluated by IPA, 6 master regulators, including MYC, Rb, and Cdkn2a were identified. ANOVA: analysis
of variance; GEO: gene expression omnibus; IPA: ingenuity pathway analysis

BALB/c congenic strains of mice carrying two tumor resistance alleles (Pctr1 and Pctr2) are more resistant
than mice carrying only one of the resistance alleles [Figure 2]. This led us to hypothesize that drug
combinations targeting these pathways are likely to have a cooperative effect in inhibiting tumor cell
growth. The p16/Rb and Mtor/PI3K pathways are frequently dysregulated in both mouse plasma cell tumors
and in human multiple myeloma [18-20] .

SYNERGISTIC DRUG COMBINATION PHENOCOPIES RESISTANCE ALLELES
The activity of combining Mtor and histone deacetylase (HDAC) inhibitors, rapamycin and entinostat
respectively, chosen to target the mouse tumor susceptibility pathways (p16/Rb and Mtor/PI3K) was
found to be synergistic in limiting the growth of a number of B lineage tumor cell lines, including mouse
[20]
plasma cell tumors, and the human B cell neoplasms, mantle cell lymphoma, and multiple myeloma . We
found that combining rapamycin and entinostat elicited responses distinct from a simple combination or
[19]
the additive effects of the two drugs . As such, we developed a rational, unbiased approach to uncover
mechanisms of drug synergy for this combination.

Systems approach
We evaluated the synergistic activity of combining Mtor and HDAC inhibitors at the organismal, cellular,
[19]
and molecular levels with a cross-disciplinary “systems pharmacology” approach [Figure 3] . While
the future impact of these specific Mtor/HDAC findings is intrinsically linked to the outcome of clinical
investigations, there is broader potential for further application and development of our approach. The
integration of patient datasets in the identification of a core synergistic response signature offers particular
opportunities for the development of companion diagnostics to aid in the clinical development of these
combinations. Gene expression-based signatures of cooperative drug responses may prove beneficial for
pre-treatment stratification of patients most likely to benefit from a particular drug combination, or as
an early response biomarker specific for the combination response and intrinsically linked to expression