Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. METHODS: The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. RESULTS: EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. CONCLUSIONS: Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Molecular, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia.

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations affect outcomes of patients treated with intensive chemotherapy. We studied 1725 patients newly diagnosed with AML (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (ie, FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes, providing a rationale to study the biology and treatment approaches in this molecular group. This trial was registered at www.clinicaltrials.gov as #NCT00048958 (CALGB 8461), #NCT00899223 (CALGB 9665), and #NCT00900224 (CALGB 20202).

TP-0903 Is Active in Preclinical Models of Acute Myeloid Leukemia with TP53 Mutation/Deletion.

Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 3-year overall survival of <5%. While inhibition of kinases involved in cell cycle regulation induces synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP-0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, Chk1/2, and other cell cycle regulators. Here, we evaluated the preclinical activity of TP-0903 in TP53 mutant AML cell lines, including a single-cell clone of MV4-11 containing a TP53 mutation (R248W), Kasumi-1 (R248Q), and HL-60 (TP 53 null). TP-0903 inhibited cell viability (IC50, 12−32 nM) and induced apoptosis at 50 nM. By immunoblot, 50 nM TP-0903 upregulated pChk1/2 and pH2AX, suggesting induction of DNA damage. The combination of TP-0903 and decitabine was additive in vitro, and in vivo significantly prolonged median survival compared to single-agent treatments in mice xenografted with HL-60 (vehicle, 46 days; decitabine, 55 days; TP-0903, 63 days; combination, 75 days) or MV4-11 (R248W) (51 days; 62 days; 81 days; 89 days) (p < 0.001). Together, these results provide scientific premise for the clinical evaluation of TP-0903 in combination with decitabine in TP53 mutant AML.

Antioxidant and pro-oxidant mechanisms of (+) catechin in microsomal CYP2E1-dependent oxidative stress.

The objectives of this work were to evaluate the effects of catechin on cytochrome P450 2E1 (CYP2E1)-dependent oxidative stress. Microsomes co-expressing human CYP2E1 with NADPH cytochrome P450 reductase and cytochrome b5 were incubated with NADPH and DTPA at pH 7.0. Superoxide anion generation was specifically detected by spin-trapping with DEPMPO. Generation of the DEPMPO-OOH adduct was not observed in the absence of CYP2E1 and in the presence of superoxide dismutase (SOD) or catechin, while catalase was ineffective. Reactive oxygen species generation was detected with 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) by the EPR-detection of its oxidation product, 3-carboxy-proxyl radical (CP●). CP● generation was not observed in the absence of CYP2E1 and in the presence of SOD, while catalase was ineffective. In contrast, catechin increased CPH oxidation, an effect that was not observed in the absence of CYP2E1 or in the presence of SOD (but not catalase), and was not associated with an increase in oxygen consumption. Catechin also increased the non-specific oxidation of the probes CPH and hydroethidine by the superoxide anion-generating system xanthine plus xanthine oxidase. Catechin oxidized CPH in the presence of horseradish peroxidase plus hydrogen peroxide, a catechin radical-generating system. In conclusion, catechin exhibits both antioxidant (superoxide-scavenging) and pro-oxidant effects under CYP2E1-dependent oxidative stress.

The long noncoding RNA, treRNA, decreases DNA damage and is associated with poor response to chemotherapy in chronic lymphocytic leukemia.

The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have investigated lncRNAs in chronic lymphocytic leukemia (CLL). We have identified one particular lncRNA, treRNA, which is overexpressed in CLL B-cells. We measured transcript expression in 144 CLL patient samples and separated samples into high or low expression of treRNA relative to the overall median. We found that high expression of treRNA is significantly associated with shorter time to treatment. High treRNA also correlates with poor prognostic indicators such as unmutated IGHV and high ZAP70 protein expression. We validated these initial findings in samples collected in a clinical trial comparing the nucleoside analog fludarabine alone or in combination with the alkylating agent cyclophosphamide in untreated CLL samples collected prior to starting therapy (E2997). High expression of treRNA was independently prognostic for shorter progression free survival in patients receiving fludarabine plus cyclophosphamide. Given these results, in order to study the role of treRNA in DNA damage response we generated a model cell line system where treRNA was over-expressed in the human B-CLL cell line OSU-CLL. Relative to the vector control line, there was less cell death in OSU-CLL over-expressing treRNA after exposure to fludarabine and mafosfamide, due in part to a reduction in DNA damage. Therefore, we suggest that treRNA is a novel biomarker in CLL associated with aggressive disease and poor response to chemotherapy through enhanced protection against cytotoxic mediated DNA damage.