Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3.

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Identification of a highly efficient dual type I/II FMS-like tyrosine kinase inhibitor that disrupts the growth of leukemic cells.

Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.

Deficiency of Antioxidative Paraoxonase 2 (Pon2) Leads to Increased Number of Phenotypic LT-HSCs and Disturbed Erythropoiesis.

BACKGROUND: Long-term hematopoietic stem cells (LT-HSCs) reside in bone marrow niches with tightly controlled reactive oxygen species (ROS) levels. ROS increase results into LT-HSC differentiation and stem cell exhaustion. Paraoxonase 2 (PON2) has been shown to be important for ROS control. OBJECTIVES: We investigate the effects of inactivation of the PON2 gene on hematopoietic cell differentiation and activity. METHODS AND RESULTS: In young mice with inactivated Pon2 gene (Pon2 -/-, <3 months), we observed an increase of LT-HSCs and a reduced frequency of progenitor cells. In competitive transplantations, young Pon2-/- BM outcompeted WT BM at early time points. ROS levels were significantly increased in Pon2-/- whole BM, but not in Pon2-/- LT-HSCs. In more differentiated stages of hematopoiesis, Pon2 deficiency led to a misbalanced erythropoiesis both in physiologic and stress conditions. In older mice (>9 months), Pon2 depletion caused an increase in LT-HSCs as well as increased levels of granulocyte/macrophage progenitors (GMPs) and myeloid skewing, indicating a premature aging phenotype. No significant changes in ROS levels in old Pon2-/- LT- and short-term (ST-) HSCs were observed, but a significant reduction of spontaneous apoptotic cell death was measured. RNA-seq analysis in Pon2 -/- LT-HSCs identified overrepresentation of genes involved in the C-X-C chemokine receptor type 4 (Cxcr4) signaling, suggesting compensatory mechanisms to overcome ROS-mediated accelerated aging in hematopoietic progenitor cells. CONCLUSIONS: In summary, our current data indicate that PON2 is involved in the regulation of HSC functions.

Definitive evidence for Club cells as progenitors for mutant Kras/Trp53-deficient lung cancer.

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide definitive evidence for Club cells as progenitors for LUAD. Using in vivo lineage tracing, we find that a subset of Kras12V -expressing and Trp53-deficient Club cells act as precursors for LUAD and we define the stepwise trajectory of Club cell-initiated tumors leading to lineage marker conversion and aggressive LUAD. Our results establish Club cells as cells-of-origin for LUAD and demonstrate that Club cell-initiated tumors have the potential to develop aggressive LUAD.

NCAM1 (CD56) promotes leukemogenesis and confers drug resistance in AML.

Neural cell adhesion molecule 1 (NCAM1; CD56) is expressed in up to 20% of acute myeloid leukemia (AML) patients. NCAM1 is widely used as a marker of minimal residual disease; however, the biological function of NCAM1 in AML remains elusive. In this study, we investigated the impact of NCAM1 expression on leukemogenesis, drug resistance, and its role as a biomarker to guide therapy. Beside t(8;21) leukemia, NCAM1 expression was found in most molecular AML subgroups at highly heterogeneous expression levels. Using complementary genetic strategies, we demonstrated an essential role of NCAM1 in the regulation of cell survival and stress resistance. Perturbation of NCAM1 induced cell death or differentiation and sensitized leukemic blasts toward genotoxic agents in vitro and in vivo. Furthermore, Ncam1 was highly expressed in leukemic progenitor cells in a murine leukemia model, and genetic depletion of Ncam1 prolonged disease latency and significantly reduced leukemia-initiating cells upon serial transplantation. To further analyze the mechanism of the NCAM1-associated phenotype, we performed phosphoproteomics and transcriptomics in different AML cell lines. NCAM1 expression strongly associated with constitutive activation of the MAPK-signaling pathway, regulation of apoptosis, or glycolysis. Pharmacological inhibition of MEK1/2 specifically inhibited proliferation and sensitized NCAM1+ AML cells to chemotherapy. In summary, our data demonstrate that aberrant expression of NCAM1 is involved in the maintenance of leukemic stem cells and confers stress resistance, likely due to activation of the MAPK pathway. Targeting MEK1/2 sensitizes AML blasts to genotoxic agents, indicating a role for NCAM1 as a biomarker to guide AML treatment.