Grb7 Upregulation Is a Molecular Adaptation to HER2 Signaling Inhibition Due to Removal of Akt-Mediated Gene Repression

The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2+ breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs

The new small tyrosine-kinase inhibitor ARQ531 targets acute myeloid leukemia cells by disrupting multiple tumor-addicted programs

Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia

Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders

Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD+-independent HDACs are an established therapeutic target, the relevance of NAD+-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited

Fasting potentiates the anticancer activity of tyrosin kinase inhibitors by strenghtening MAPK signalling inhibition.

Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use

EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

Cell-cycle analysis andClick-iTdetection of RNA and Protein synthesis. A) Cell-cycle analysis with PI staining of the nuclei after 48 h of treatment. Overnight serum starvation is shown as a positive control of induced cell cycle synchronization in G0/G1 phase. Cell phase analysis was done with ModFit LT 3.2 software by using the Sync Wizard model (30000 cells/sample in biological duplicate). B) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 5 μM Actinomycin D, an RNA synthesis blocking agent, then subjected to Click-it biochemistry and flow-cytometry analyses including 7-AAD to identify living cells. C) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 350 μM Cycloheximide, as a positive control for protein synthesis inhibition, then stained as in B. In B and C. Experiments were carried out on two biological replicates (50000 events/sample). (PDF 1562 kb