EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). METHODS: We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). RESULTS: eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. CONCLUSIONS: We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Defining the role of prolactin as an invasion suppressor hormone in breast cancer cells.

Prolactin hormone (PRL) is well characterized as a terminal differentiation factor for mammary epithelial cells and as an autocrine growth/survival factor in breast cancer cells. However, this function of PRL may not fully signify its role in breast tumorigenesis. Cancer is a complex multistep progressive disease resulting not only from defects in cell growth but also in cell differentiation. Indeed, dedifferentiation of tumor cells is now recognized as a crucial event in invasion and metastasis. PRL plays a critical role in inducing/maintaining differentiation of mammary epithelial cells, suggesting that PRL signaling could serve to inhibit tumor progression. We show here that in breast cancer cells, PRL and Janus-activated kinase 2, a major kinase involved in PRL signaling, play a critical role in regulating epithelial-mesenchymal transformation (EMT), an essential process associated with tumor metastasis. Activation of the PRL receptor (PRLR), achieved by restoring PRL/JAK2 signaling in mesenchymal-like breast cancer cells, MDA-MB-231, suppressed their mesenchymal properties and reduced their invasive behavior. While blocking PRL autocrine function in epithelial-like breast cancer cells, T47D, using pharmacologic and genetic approaches induced mesenchymal-like phenotypic changes and enhanced their invasive propensity. Moreover, our results indicate that blocking PRL signaling led to activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and transforming growth factor-beta/Smad signaling pathways, two major prometastatic pathways. Furthermore, our results indicate that following PRL/JAK2 inhibition, ERK1/2 activation precedes and is required for Smad2 activation and EMT induction in breast cancer cells. Together, these results highlight PRL as a critical regulator of epithelial plasticity and implicate PRL as an invasion suppressor hormone in breast cancer.

Endoplasmic reticulum association and N-linked glycosylation of the human Nrf3 transcription factor.

We have analysed the molecular and cellular regulation of the basic-leucine zipper (bZIP) transcription factor Nrf3 (NFE2-Related Factor 3). Cycloheximide studies revealed a rapid turnover of Nrf3. We showed that the proteasome inhibitor MG-132 increases Nrf3 protein levels. Furthermore, we demonstrated that Nrf3 is an N-glycosylated protein associated with the endoplasmic reticulum. Thus, our studies provide the first evidence of a post-translational modification of Nrf3.

Recurrent familial hypocalcemia due to germline mosaicism for an activating mutation of the calcium-sensing receptor gene.

De novo activating mutations in the calcium-sensing receptor (CASR) gene are a common cause of sporadic isolated hypoparathyroidism. Here, we describe a family in which two affected siblings were found to be heterozygous for a novel F788L mutation in the fifth transmembrane domain encoded by exon 7 of the CASR. Both parents and the third sibling were clinically unaffected and genotypically normal by direct sequencing of their leukocyte exon 7 PCR amplicons. However, the mother was revealed to be a mosaic for the mutation by sequence analysis of multiple subclones as well as denaturing HPLC of the CASR exon 7 leukocyte PCR product. A functional analysis of the mutation was performed by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. The mutant CASR was expressed at a similar level as the wild type. The F788L mutant produced a significant shift to the left relative to the wild-type CASR in the MAPK response to increasing extracellular calcium concentrations. This is the first report of mosaicism for an activating CASR mutation and suggests that care should be exercised in counseling for risks of recurrence in a situation where a de novo mutation appears likely.

Abnormal differentiation of erythroid precursors in p45 NF-E2(-/-) mice.

The transcription factor p45 nuclear factor-erythroid-derived 2 (NF-E2) plays major roles in erythroid and megakaryocytic lineages. Here, we investigated the role of p45 NF-E2 in erythroid differentiation in vivo. Absence of p45 NF-E2 in mice leads to a twofold increase in serum erythropoietin levels. In the bone marrow of these animals, we found a different distribution of precursor populations compared to wild-type mice, suggesting abnormal differentiation. Loss of p45 NF-E2 was also associated with an increase in splenic erythropoiesis, as evidenced by an accumulation of early precursors, namely, late basophilic and polychromatic erythroblasts. These observations are consistent with a stress erythropoiesis phenotype and indicate that the spleen is likely compensating for ineffective erythropoiesis in the bone marrow. Analysis of bone marrow samples revealed increased GATA1 levels, as well as an increased proportion of erythroid cells arrested at the G(1) stage of cell cycle in p45 NF-E2-deficient mice. These results suggest that p45 NF-E2 is required for the differentiation of erythroid precursors.

Nrf3-deficient mice are not protected against acute lung and adipose tissue damages induced by butylated hydroxytoluene.

We found that both wild type and Nrf3 (NF-E2-related factor 3) deficient mice are sensitive to BHT single administration exhibiting respiratory distress and considerably lose body weight following treatment. At time of sacrifice, the BHT-treated Nrf3-/- mice had lost significantly more body weight than their WT counterparts. In the lung, transcript levels of the transcription factors Nrf1, Nrf2 and Nrf3 were differentially regulated by BHT treatment. In addition, genes implicated in adipogenesis were repressed following BHT exposure in the white adipose tissue. Together, our data provide the first evidence that BHT exposure not only affects lung function but also leads to impaired adipogenesis in adipocytes.

SHP-2 regulates SOCS-1-mediated Janus kinase-2 ubiquitination/degradation downstream of the prolactin receptor.

The protein tyrosine phosphatase SHP-2 is an important regulator of the Janus kinase-2 (Jak2)/signal transducer and activator of transcription (Stat) pathway downstream of the cytokine/prolactin receptor family. We report that SHP-2 dephosphorylates tyrosine (Tyr-1007) of Jak2 kinase, a critical recruitment site for the ubiquitin ligase-associated inhibitory protein suppressor of cytokine signaling-1 (SOCS-1), thereby contributing to Jak2 stability. Inactivation of SHP-2 function by blocking receptor/SHP-2 association or by using a catalytically inactive mutant of SHP-2 led to a marked increase in Jak2 ubiquitination/degradation, Jak2 phosphorylation on Tyr-1007, and Jak2/SOCS-1 association. Furthermore, functional studies indicate that modulating the interaction of Jak2/SOCS-1 by SHP-2 is essential for prolactin/Stat5-mediated signaling. Together our results provide a novel function for SHP-2 as a positive regulator of cytokine receptor signaling by regulating ubiquitination/degradation pathways.