Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.

Multiple myeloma cells secrete more disulfide bond-rich proteins than any other mammalian cell. Thus, inhibition of protein disulfide isomerases (PDI) required for protein folding in the endoplasmic reticulum (ER) should increase ER stress beyond repair in this incurable cancer. Here, we report the mechanistically unbiased discovery of a novel PDI-inhibiting compound with antimyeloma activity. We screened a 30,355 small-molecule library using a multilayered multiple myeloma cell-based cytotoxicity assay that modeled disease niche, normal liver, kidney, and bone marrow. CCF642, a bone marrowsparing compound, exhibited a submicromolar IC 50 in 10 of 10 multiple myeloma cell lines. An active biotinylated analog of CCF642 defined binding to the PDI isoenzymes A1, A3, and A4 in MM cells. In vitro, CCF642 inhibited PDI reductase activity about 100-fold more potently than the structurally distinct established inhibitors PACMA 31 and LOC14. Computational modeling suggested a novel covalent binding mode in activesite CGHCK motifs. Remarkably, without any further chemistry optimization, CCF642 displayed potent efficacy in an aggressive syngeneic mouse model of multiple myeloma and prolonged the lifespan of C57BL/KaLwRij mice engrafted with 5TGM1-luc myeloma, an effect comparable to the first-line multiple myeloma therapeutic bortezomib. Consistent with PDI inhibition, CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis-inducing calcium release. Overall, our results provide an illustration of the utility of simple in vivo simulations as part of a drug discovery effort, along with a sound preclinical rationale to develop a new small-molecule therapeutic to treat multiple myeloma.

Topoisomerases alter DNA topology and are vital for the maintenance of genomic integrity. Topoisomerases I and II are also targets for widely used antitumor agents. We demonstrated previously that in the human leukemia cell line, HL-60, resistance to topoisomerase (topo) II-targeting drugs such as etoposide is associated with site-specific hypophosphorylation of topo II␣. This effect can be mimicked in sensitive cells treated with the intracellular Ca 2؉ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid (BAPTA-AM). Here we identify Ser-1106 as a major phosphorylation site in the catalytic domain of topo II␣. This site lies within the consensus sequence for the acidotrophic kinases, casein kinase I and casein kinase II. Mutation of serine 1106 to alanine (S1106A) abrogates phosphorylation of phosphopeptides that were found to be hypophosphorylated in resistant HL-60 cells or sensitive cells treated with BAPTA-AM. Purified topo II␣ containing a S1106A substitution is 4-fold less active than wild type topo II␣ in decatenating kinetoplast DNA and also exhibits a 2-4-fold decrease in the level of etoposidestabilized DNA cleavable complex formation. Saccharomyces cerevisiae (JN394t2-4) cells expressing S1106A mutant topo II␣ protein are more resistant to the cytotoxic effects of etoposide or amsacrine. These results demonstrate that Ca 2؉ -regulated phosphorylation of Ser-1106 in the catalytic domain of topo II␣ modulates the enzymatic activity of this protein and sensitivity to topo II-targeting drugs.Topoisomerases alter DNA topology for the efficient processing of genetic material (1-3). These enzymes play a pivotal role in the maintenance of genomic integrity and are essential for many chromosomal functions including DNA replication and recombination, transcription, and chromosome segregation (1-3). Topoisomerases regulate various cellular processes by relaxing and untangling the intertwined strands of DNA. Two major categories of topoisomerases, type I and type II, have been characterized. The type II enzymes, which consist of two highly homologous isoforms in humans, topoisomerase (topo) 1 II␣ and topo II␤ with molecular masses of 170 and 180 kDa, respectively, catalyze the ATP-dependent transport of one intact DNA double helix through another by creating a transient double-stranded break (1).The essential nature of topo II␣ during cell proliferation and its ability to cleave DNA in a reversible manner makes topo II␣ an ideal target for agents that poison the enzyme (4, 5). In the presence of DNA-damaging topo II-targeting drugs, topo II is converted to a nuclease that irreversibly cleaves duplex DNA. The "poisoning" of the enzyme is via the trapping of the transient reaction intermediate, termed a cleavable complex, which is composed of topo II bound covalently to the 5Ј end of the cleaved DNA strands. This leads to DNA damage, apoptosis, genomic instability, and cell death. Several clinically effective cancer chemotherapeutic agents, daunorubicin, doxorubicin (DOX), amsacrine (m-AMSA), and eto...

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerasetargeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent.

TET2 is frequently mutated in myeloid neoplasms. Genetic TET2 deficiency leads to skewed myeloid differentiation and clonal expansion, but minimal residual TET activity is critical for survival of neoplastic progenitor and stem cells. Consistent with mutual exclusivity of TET2 and neomorphic IDH1/2 mutations, here we report that IDH1/2 mutant–derived 2-hydroxyglutarate is synthetically lethal to TET dioxygenase–deficient cells. In addition, a TET-selective small-molecule inhibitor decreases cytosine hydroxymethylation and restricted clonal outgrowth of TET2 mutant but not normal hematopoietic precursor cells in vitro and in vivo. Although TET inhibitor phenocopied somatic TET2 mutations, its pharmacologic effects on normal stem cells are, unlike mutations, reversible. Treatment with TET inhibitor suppresses the clonal evolution of TET2-mutant cells in murine models and TET2-mutated human leukemia xenografts. These results suggest that TET inhibitors may constitute a new class of targeted agents in TET2-mutant neoplasia. Significance: Loss-of-function somatic TET2 mutations are among the most frequent lesions in myeloid neoplasms and associated disorders. Here we report a strategy for selective targeting of residual TET dioxygenase activity in TET-deficient clones that results in restriction of clonal evolution in vitro and in vivo. See related video: https://aacrjournals.org/webinar-minimal-tet-activity-targetable-vulnerability-tet2-and-neomorphic-idh12-mutant