Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

Chronic Lymphocytic Leukemia (CLL) is an incurable disease, characterized by the accumulation of malignant B-lymphocytes in the blood stream (quiescent state) and homing tissues (where they can proliferate). In CLL, the targeting of B-cell receptor signaling through a Burton's tyrosine kinase inhibitor (ibrutinib) has rendered outstanding clinical results. However, complete remission is not guaranteed due to drug resistance or relapse, revealing the need for novel approaches for CLL treatment. The characterization of metabolic rewiring in proliferative cancer cells is already being applied for diagnostic and therapeutic purposes, but our knowledge of quiescent cell metabolism—relevant for CLL cells—is still fragmentary. Recently, we reported that glutamine metabolism in primary CLL cells bearing the del11q deletion is different from their del11q negative counterparts, making del11q cells especially sensitive to glutaminase and glycolysis inhibitors. In this work, we used our primary CLL lymphocyte bank and compounds interfering with central carbon metabolism to define metabolic traits associated with ibrutinib resistance. We observe a differential basal metabolite uptake linked to ibrutinib resistance, favoring glutamine uptake and catabolism. Upon ibrutinib treatment, the redox balance in ibrutinib resistant cells is shifted toward NADPH accumulation, without an increase in glutamine uptake, suggesting alternative metabolic rewiring such as the activation of fatty acid oxidation. In accordance to this idea, the curtailing of fatty acid oxidation by CPT1 inhibition (etomoxir) re-sensitized resistant cells to ibrutinib. Our results suggest that fatty acid oxidation could be explored as a target to overcome ibrutinib resistance

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

BACKGROUND. Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. METHODOLOGY/PRINCIPAL FINDINGS. Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. CONCLUSIONS/SIGNIFICANCE. Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.Canadian Institutes of Health Research (16512, Cancer Consortium Training Grant Award, CancerConsortium Training Grant Award); US Lymphoma Foundation Award; National Institute of Health (RO1 GM073855); National Crime Information Center (017099); Cole Foundation Awar

Magnesium-sensitive upstream ORF controls PRL phosphatase expression to mediate energy metabolism

The phosphatases of regenerative liver (PRL) have been shown to interact with the CNNM magnesium transport regulators. Through this protein complex, PRL controls the levels of intracellular magnesium. Our study uncovers a remarkable posttranscriptional feedback mechanism by which magnesium controls PRL expression in mammalian cells. Here we show that regulation of PRL mRNA translation by magnesium depends on a 5'UTR-located upstream ORF, which is conserved among all vertebrates, proposing an evolutionary molecular mechanism of action by a divalent ion. This magnesium-sensing mechanism, which also involves the key metabolic sensor AMPK, is thus central to maintain cellular homeostasis in mammalian cells.Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5'UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics

Regulation of eukaryotic initiation factor 4AII by MyoD during murine myogenic cell differentiation.

Gene expression during muscle cell differentiation is tightly regulated at multiple levels, including translation initiation. The PI3K/mTOR signalling pathway exerts control over protein synthesis by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that stimulates recruitment of ribosomes to mRNA templates. One of the subunits of eIF4F, eIF4A, supplies essential helicase function during this phase of translation. The presence of two cellular eIF4A isoforms, eIF4AI and eIF4AII, has long thought to impart equivalent functions to eIF4F. However, recent experiments have alluded to distinct activities between them. Herein, we characterize distinct regulatory mechanisms between the eIF4A isoforms during muscle cell differentiation. We find that eIF4AI levels decrease during differentiation whereas eIF4AII levels increase during myofiber formation in a MyoD-dependent manner. This study characterizes a previously undefined mechanism for eIF4AII regulation in differentiation and highlights functional differences between eIF4AI and eIF4AII. Finally, RNAi-mediated alterations in eIF4AI and eIF4AII levels indicate that the myogenic process can tolerate short term reductions in eIF4AI or eIF4AII levels, but not both

CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A

Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF) 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition

Transcriptional changes in eIF4AII mRNA levels during C2C12 differentiation.

<p>(<b>A</b>) Changes in eIF4AI and eIF4AII mRNA levels during C2C12 cell differentiation. mRNA levels were determined by RT-qPCR and are standardized to GAPDH levels. n = 3±SEM. (B) Transcriptional changes in eIF4AI and eIF4AII mRNA levels during primary myoblast differentiation. mRNA levels were determined by RT-qPCR and are standardized to GAPDH levels. n = 4±SEM. (<b>C</b>) Nuclear Run-On analysis of GAPDH, MyoD and eIF4AII transcription in C2C12 cells at days 0 and 3 after induction of differentiation. Probes targeting the 5′ and 3′ UTRs of eIF4AII were used to distinguish the transcript from that of eIF4AI. (<b>D</b>) Quantiation of nuclear run-on experiments. Changes in eIF4AII transcription was quantified using a Typhoon Scanner (GE Healthcare) (values are normalized to GAPDH mRNA levels which did not change over this time period).</p

Expression of eIF4AI and eIF4AII during C2C12 differentiation.

<p>(<b>A</b>) Phase contrast images of C2C12 cells grown in the presence of DM for the indicated number of days (d). Scale bars represent 50 µm. (<b>B</b>) Western Blot analysis documenting expression levels of the indicated proteins during C2C12 cell differentiation. Long (l.e.) and short (s.e.) exposures of the eIF4AII Western blot are presented. (<b>C</b>) Quantification of changes in eIF4AI and eIF4AII protein levels relative to those obtained on day 0. n = 3±SEM. (<b>D</b>) ³⁵S-methionine/cysteine incorporation into TCA-insoluble protein. C2C12 cells were induced to differentiate and protein extracts were prepared at the indicated time points. Cells were labeled for 30 min and the amount of radiolabeled protein quantitated by TCA precipitation. Values are standardized against total protein content. n = 3±SEM. (<b>E</b>) eIF4AI/II are efficiently incorporated into the eIF4F complex during C2C12 differentiation. m⁷GTP affinity purification of the eIF4F complex from C2C12 cells at the indicated days following induction of differentiation. Western blots to the indicated proteins were performed on an aliquot of input extract (lanes 1–4), GDP eluents (lanes 5–8), and m⁷GTP eluents (lanes 9–12).</p