Targeting OXPHOS de novo purine synthesis as the nexus of FLT3 inhibitor-mediated synergistic antileukemic actions.

Using a genome-wide CRISPR screen, we identified CDK9, DHODH, and PRMT5 as synthetic lethal partners with gilteritinib treatment in fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) and genetically and pharmacologically validated their roles in gilteritinib sensitivity. The presence of FLT3-ITD is associated with an increase in anaerobic glycolysis, rendering leukemia cells highly sensitive to inhibition of glycolysis. Supportive of this, our data show the enrichment of single guide RNAs targeting 28 glycolysis-related genes upon gilteritinib treatment, suggesting that switching from glycolysis to oxidative phosphorylation (OXPHOS) may represent a metabolic adaption of AML in gilteritinib resistance. CDK9i/FLT3i, DHODHi/FLT3i, and PRMT5i/FLT3i pairs mechanistically converge on OXPHOS and purine biosynthesis blockade, implying that targeting the metabolic functions of these three genes and/or proteins may represent attractive strategies to sensitize AML to gilteritinib treatment. Our findings provide the basis for maximizing therapeutic impact of FLT3-ITD inhibitors and a rationale for a clinical trial of these novel combinations.

Selinexor Combined with Ibrutinib Demonstrates Tolerability and Safety in Advanced B-Cell Malignancies: A Phase I Study.

PURPOSE: Dual blockade of Bruton's tyrosine kinase with ibrutinib and selinexor has potential to deepen responses for patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: In this phase I study (clinicaltrials.gov: NCT02303392), adult patients with CLL/NHL, relapsed/refractory to ≥1 prior therapy were enrolled. Patients received weekly oral selinexor and daily oral ibrutinib in 28-day cycles until progression or intolerance. Primary objective was to determine MTD. RESULTS: Included patients had CLL (n = 16) or NHL (n = 18; 9 Richter transformation, 6 diffuse large B-cell lymphoma, and 3 mantle cell lymphoma). Median prior therapies were 4 (range = 1-14) and 59% previously received ibrutinib. The established MTD was 40 mg of selinexor (days 1, 8, 15) and 420 mg daily ibrutinib. Common nonhematologic adverse events were fatigue (56%), nausea (53%), anorexia (41%), and diarrhea (41%) and were mostly low grade. Overall response rate was 32%. An additional 47% achieved stable disease (SD), some prolonged (up to 36 months). Median progression-free survival for patients with CLL and NHL was 8.9 [95% confidence interval (CI), 3.9-16.1] and 2.7 (95% CI, 0.7-5.4) months, respectively. For patients with CLL who did not receive prior ibrutinib, only 20% (1/5) progressed. Estimated 2-year overall survival was 73.7% (95% CI, 44.1-89.2) and 27.8% (95% CI, 10.1-48.9) for patients with CLL and NHL, respectively. CONCLUSIONS: The selinexor and ibrutinib combination has demonstrated tolerability in patients with relapsed/refractory CLL/NHL. Responses were durable. Notable responses were seen in patients with CLL with minimal prior therapy. Future study of this combination will focus on efforts to deepen remissions in patients with CLL receiving ibrutinib therapy.

Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition.

PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.

Synergistic effect of BCL2 and FLT3 co-inhibition in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.

Cotargeting of XPO1 Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematopoietic stem-cell-derived leukemia with often successive derived driver mutations. Late onset acquisition of internal tandem duplication in FLT3 (FLT3-ITD) at a high variant allele frequency often contributes to full transformation to a highly proliferative, rapidly progressive disease with poor outcome. The FLT3-ITD mutation is targetable with approved FLT3 small molecule inhibitors, including midostaurin and gilteritinib. However, outside of patients receiving allogeneic transplant, most patients fail to respond or relapse, suggesting alternative approaches of therapy will be required. We employed genome-wide pooled CRISPR knockout screening as a method for large-scale identification of targets whose knockout produces a phenotypic effect that enhances the antitumor properties of FLT3 inhibitors. Among the candidate targets we identified the effect of XPO1 knockout to be synergistic with midostaurin treatment. Next, we validated the genetic finding with pharmacologic combination of the slowly reversible XPO1 inhibitor selinexor with midostaurin and gilteritinib in FLT3-ITD AML cell lines and primary patient samples. Lastly, we demonstrated improved survival with either combination therapy compared to its monotherapy components in an aggressive AML murine model, supporting further evaluation and rapid clinical translation of this combination strategy.

Factors related to Secchi depths and their stability over time as determined from a probability sample of US lakes.

A probabilistic sample of lakes in the 48 coterminous US lakes was made by the United States Environmental Protection Agency in the 2007 National Lakes Assessment. Because of the statistical design, the results of our analyses of Secchi depths (SD) apply to a population of 45,265 lakes. We found statistically significant differences in mean Secchi depths between natural (1.57 m) and man-made lakes (1.18 m). The most important variable correlated with SD was turbidity, an optical measure related to suspended particles in the water column. For most lakes, chlorophyll a was highly correlated with both turbidity and SD, but several lakes had more turbidity and lower SD than expected based on chlorophyll a alone, indicating that non-algal suspended solids were an important factor. On an ecoregion basis, the non-algal suspended solids in the lake waters were related to the average levels of suspended solids in streams located in that ecoregion, and the non-algal suspended solids were more important in man-made than natural lakes. Phosphorus and nitrogen were directly correlated with chlorophyll a and turbidity and inversely correlated with SD. Based on diatom-inferred Secchi depths for the tops and bottoms of sediment cores from lakes in Ecoregions VIII and VII (excluding lakes in Minnesota) representing 40% of the natural lakes in the US, there has been no decrease in water transparency in that population of lakes in the past 70 or more years when the US population increased by 134%. We do not have information to determine if the other 60% of lakes have or have not changed.