Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.

Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo . Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo . Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

MicroRNA let-7b downregulates AML1-ETO oncogene expression in t(8;21) AML by targeting its 3'UTR.

BACKGROUND: Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) chromosomal translocation is among the most common subtypes of AML and produces the AML1-ETO (RUNX1-ETO, RUNX1-RUNX1T1) oncogenic fusion gene. AML1-ETO functions as an aberrant transcription factor which plays a key role in blocking normal hematopoiesis. Thus, the expression of AML1-ETO is critical to t(8;21) AML leukemogenesis and maintenance. Post-transcriptional regulation of gene expression is often mediated through interactions between trans-factors and cis-elements within transcript 3'-untranslated regions (UTR). AML1-ETO uses the 3'UTR of the ETO gene, which is not normally expressed in hematopoietic cells. Therefore, the mechanisms regulating AML1-ETO expression via the 3'UTR are attractive therapeutic targets. METHODS: We used RNA-sequencing of t(8;21) patients and cell lines to examine the 3'UTR isoforms used by AML1-ETO transcripts. Using luciferase assay approaches, we test the relative contribution of 3'UTR cis elements to AML1-ETO expression. We further use let-7b microRNA mimics and anti-let-7b sponges for functional studies of t(8;21) AML cell lines. RESULTS: In this study, we examine the regulation of AML1-ETO via the 3'UTR. We demonstrate that AML1-ETO transcripts primarily use a 3.7 kb isoform of the ETO 3'UTR in both t(8;21) patients and cell lines. We identify a negative regulatory element within the AML1-ETO 3'UTR. We further demonstrate that the let-7b microRNA directly represses AML1-ETO through this site. Finally, we find that let-7b inhibits the proliferation of t(8;21) AML cell lines, rescues expression of AML1-ETO target genes, and promotes differentiation. CONCLUSIONS: AML1-ETO is post-transcriptionally regulated by let-7b, which contributes to the leukemic phenotype of t(8;21) AML and may be important for t(8;21) leukemogenesis and maintenance.

VX-322: a novel dual receptor tyrosine kinase inhibitor for the treatment of acute myelogenous leukemia.

In acute myelogenous leukemia (AML), the FLT3 receptor tyrosine kinase (RTK) is highly expressed with 30% of patients expressing a mutated, constitutively active form of this protein. To inhibit this receptor, VX-322 was developed and found to be very potent against both the FLT3 and c-KIT RTKs with enzyme K(i) values of <1 nM and a cellular IC(50) between 1 and 5 nM. It was efficacious in a FLT3-ITD dependent myeloproliferative mouse model, doubling survival compared to other FLT3 inhibitors, with 25% of the mice cured. Upon treatment of primary AML patient blast cells, the dual inhibition of FLT3 and c-KIT was superior to inhibitors targeting a single RTK. Thus, this compound may represent an improved pharmacologic and selectivity profile that could be effective in the treatment of AML.