A loss-of-function genetic screening reveals synergistic targeting of AKT/mTOR and WTN/ß-catenin pathways for treatment of AML with high PRL-3 phosphatase.

BACKGROUND: Protein tyrosine phosphatase of regenerating liver 3 (PRL-3) is overexpressed in a subset of AML patients with inferior prognosis, representing an attractive therapeutic target. However, due to relatively shallow pocket of the catalytic site of PRL-3, it is difficult to develop selective small molecule inhibitor. METHODS: In this study, we performed whole-genome lentiviral shRNA library screening to discover synthetic lethal target to PRL-3 in AML. We used specific small molecule inhibitors to validate the synthetic lethality in human PRL-3 high vs PRL-3 low human AML cell lines and primary bone marrow cells from AML patients. AML mouse xenograft model was used to examine the in vivo synergism. RESULTS: The list of genes depleted in TF1-hPRL3 cells was particularly enriched for members involved in WNT/ß-catenin pathway and AKT/mTOR signaling. These findings prompted us to explore the impact of AKT/mTOR signaling inhibition in PRL-3 high AML cells in combination with WNT/ß-catenin inhibitor. VS-5584, a novel, highly selective dual PI3K/mTOR inhibitor, and ICG-001, a WNT inhibitor, were used as a combination therapy. A synthetic lethal interaction between mTOR/AKT pathway inhibition and WNT/ß-catenin was validated by a variety of cellular assays. Notably, we found that treatment with these two drugs significantly reduced leukemic burden and prolonged survival of mice transplanted with human PRL-3 high AML cells, but not with PRL-3 low AML cells. CONCLUSIONS: In summary, our results support the existence of cooperative signaling networks between AKT/mTOR and WNT/ß-catenin pathways in PRL-3 high AML cells. Simultaneous inhibition of these two pathways could achieve robust clinical efficacy for this subtype of AML patient with high PRL-3 expression and warrant further clinical investigation.

NF-κB promotes the stem-like properties of leukemia cells by activation of LIN28B.

AIM: To examine whether nuclear factor kappa B (NF-κB) activity regulates LIN28B expression and their roles in leukemia stem cell (LSC)-like properties. METHODS: We used pharmacological inhibitor and cell viability assays to examine the relation between NF-κB and LIN28B. Western blot and qRT-PCR was employed to determine their protein and mRNA levels. Luciferase reporter was constructed and applied to explore the transcriptional regulation of LIN28B. We manipulated LIN28B level in acute myeloid leukemia (AML) cells and investigated LSC-like properties with colony forming and serial replating assays. RESULTS: This study revealed the relationship between NF-κB and LIN28B in AML cells through drug inhibition and overexpression experiments. Notably, inhibition of NF-κB by pharmacological inhibitors reduced LIN28B expression and decreased cell proliferation. We demonstrated that NF-κB binds to the -819 to -811 region of LIN28B promoter, and transcriptionally regulates LIN28B expression. LIN28B protein was significantly elevated in NFκB1 transfected cells compared to vector control. Importantly, ectopic expression of LIN28B partially rescued the self-renewal capacity impaired by pharmacological inhibition of NF-κB activity. CONCLUSION: These results uncover a regulatory signaling, NF-κB/LIN28B, which plays a pivotal role in leukemia stem cell-like properties and it could serve as a promising intervening target for effective treatment of AML disease.

Inhibition of LIN28B impairs leukemia cell growth and metabolism in acute myeloid leukemia.

BACKGROUND: Current conventional chemotherapy for acute myeloid leukemia (AML) can achieve remission in over 70% of patients, but a majority of them will relapse within 5 years despite continued treatment. The relapse is postulated to be due to leukemia stem cells (LSCs), which are different from normal hematopoietic stem cells (HSCs). LIN28B is microRNA regulator and stem cell reprogramming factor. Overexpression of LIN28B has been associated with advance human malignancies and cancer stem cells (CSCs), including AML. However, the molecular mechanism by which LIN28B contributes to the development of AML remains largely elusive. METHODS: We modulated LIN28B expression in AML and non-leukemic cells and investigated functional consequences in cell proliferation, cell cycle, and colony-forming assays. We performed a microarray-based analysis for LIN28B-silencing cells and interrogated gene expression data with different bioinformatic tools. AML mouse xenograft model was used to examine the in vivo function of LIN28B. RESULTS: We demonstrated that targeting LIN28B in AML cells resulted in cell cycle arrest, inhibition of cell proliferation and colony formation, which was induced by de-repression of let-7a miRNA. On the other hand, overexpression of LIN28B promoted cell proliferation. Data point to a mechanism where that inhibition of LIN28B induces metabolic changes in AML cells. IGF2BP1 was confirmed to be a novel downstream target of LIN28B via let-7 miRNA in AML. Notably, ectopic expression of LIN28B increased tumorigenicity, while silencing LIN28B led to slow tumor growth in vivo. CONCLUSIONS: In sum, these results uncover a novel mechanism of an important regulatory signaling, LIN28B/let-7/IGF2BP1, in leukemogenesis and provide a rationale to target this pathway as effective therapeutic strategy.