The metabolism of cells regulates their sensitivity to NK cells depending on p53 status

Leukemic cells proliferate faster than non-transformed counterparts. This requires them to change their metabolism to adapt to their high growth. This change can stress cells and facilitate recognition by immune cells such as cytotoxic lymphocytes, which express the activating receptor Natural Killer G2-D (NKG2D). The tumor suppressor gene p53 regulates cell metabolism, but its role in the expression of metabolism-induced ligands, and subsequent recognition by cytotoxic lymphocytes, is unknown. We show here that dichloroacetate (DCA), which induces oxidative phosphorylation (OXPHOS) in tumor cells, induces the expression of such ligands, e.g. MICA/B, ULBP1 and ICAM-I, by a wtp53-dependent mechanism. Mutant or null p53 have the opposite effect. Conversely, DCA sensitizes only wtp53-expressing cells to cytotoxic lymphocytes, i.e. cytotoxic T lymphocytes and NK cells. In xenograft in vivo models, DCA slows down the growth of tumors with low proliferation. Treatment with DCA, monoclonal antibodies and NK cells also decreased tumors with high proliferation. Treatment of patients with DCA, or a biosimilar drug, could be a clinical option to increase the effectiveness of CAR T cell or allogeneic NK cell therapies

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

Denatonium as a Bitter Taste Receptor Agonist Modifies Transcriptomic Profile and Functions of Acute Myeloid Leukemia Cells

The contribution of cell-extrinsic factors in Acute Myeloid Leukemia (AML) generation and persistence has gained interest. Bitter taste receptors (TAS2Rs) are G protein-coupled receptors known for their primary role as a central warning signal to induce aversion toward noxious or harmful substances. Nevertheless, the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. In this study, we found that AML cells express functional TAS2Rs. We also highlighted a significant association between the modulation of some TAS2Rs and the poor-prognosis AML groups, i.e., TP53- and TET2-mutated, supporting a potential role of TAS2Rs in AML cell biology. Gene expression profile analysis showed that TAS2R activation with the prototypical agonist, denatonium benzoate, significantly modulated a number of genes involved in relevant AML cellular processes. Functional assay substantiated molecular data and indicated that denatonium reduced AML cell proliferation by inducing cell cycle arrest in G0/G1 phase or induced apoptosis via caspase cascade activation. Moreover, denatonium exposure impaired AML cell motility and migratory capacity, and inhibited cellular respiration by decreasing glucose uptake and oxidative phosphorylation. In conclusion, our results in AML cells expand the observation of cancer TAS2R expression to the setting of hematological neoplasms and shed light on a role of TAS2Rs in the extrinsic regulation of leukemia cell functions

Vinorelbine and Intermittent Cyclophosphamide Sensitize an Aggressive Myc-Driven B-Cell Lymphoma to Anti-PD-1 by an Immunological Memory Effective against Tumor Re-Challenge

We have previously shown in triple-negative breast cancer (TNBC) models that a triple therapy (TT) including intermittent cyclophosphamide (C), vinorelbine (V), and anti-PD-1 activates antigen-presenting cells (APC) and generates stem like-T cells able to control local and metastatic tumor progression. In the present manuscript, we report the generation of a highly aggressive, anti-PD-1 resistant model of a high-grade, Myc-driven B-cell non-Hodgkin’s lymphoma (NHL) that can be controlled in vivo by TT but not by other chemotherapeutic agents, including cytarabine (AraC), platinum (P), and doxorubicin (D). The immunological memory elicited in tumor-bearing mice by TT (but not by other treatments) can effectively control NHL re-challenge even at very high inoculum doses. TT re-shaped the landscape of circulating innate NK cells and adaptive immune cells, including B and T cells, and significantly reduced exhausted CD4+ and CD8+ TIM3+PD-1+ T cells in the spleens of treated mice

Denatonium as a Bitter Taste Receptor Agonist Modifies Transcriptomic Profile and Functions of Acute Myeloid Leukemia Cells.

The contribution of cell-extrinsic factors in Acute Myeloid Leukemia (AML) generation and persistence has gained interest. Bitter taste receptors (TAS2Rs) are G protein-coupled receptors known for their primary role as a central warning signal to induce aversion toward noxious or harmful substances. Nevertheless, the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. In this study, we found that AML cells express functional TAS2Rs. We also highlighted a significant association between the modulation of some TAS2Rs and the poor-prognosis AML groups, i.e., TP53- and TET2-mutated, supporting a potential role of TAS2Rs in AML cell biology. Gene expression profile analysis showed that TAS2R activation with the prototypical agonist, denatonium benzoate, significantly modulated a number of genes involved in relevant AML cellular processes. Functional assay substantiated molecular data and indicated that denatonium reduced AML cell proliferation by inducing cell cycle arrest in G0/G1 phase or induced apoptosis via caspase cascade activation. Moreover, denatonium exposure impaired AML cell motility and migratory capacity, and inhibited cellular respiration by decreasing glucose uptake and oxidative phosphorylation. In conclusion, our results in AML cells expand the observation of cancer TAS2R expression to the setting of hematological neoplasms and shed light on a role of TAS2Rs in the extrinsic regulation of leukemia cell functions.: This research was supported by: Bologna AIL (Associazione Italiana contro le Leucemie)/Bologna Brancch, FATRO/Foundation Corrado and Bruno Maria Zaini-Bologna, Fabbri1905, Regione Emilia-Romagna and University of Bologna (Young Researcher's fund of the Regione Emilia Romagna, Bando Alessandro Liberati GREREMAT-Curti to AC), Italian Association for Cancer Research grant (AIRC) IG20109 to FB. VS and MC were supported by the American Society of Haematology (ASH)/Giuseppe Bigi Memorial Award and by the University of Bologna (Alma Idea Junior Grant 2017), EA was supported by AIRC IG16812. DF was supported by AIRC fellowship for abroad-2017, Società Italiana di Ematologia (SIE) and Associazione Amici di Beat Leukemia Dr. Alessandro Cevenini ONLUSS

The pan-class I PI3 kinase inhibitor, NVP-BKM120, demonstrates anti-leukemic activity in acute myeloid leukemia

Background: Aberrant activation of PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represents a potential therapeutic approach in AML. NVPBKM120 (BKM120) is a pan-class I PI3K inhibitor which exerts anti-proliferative and cytotoxic effects on several solid tumors and hematological malignancies by inhibiting PI3K/Akt/mTOR activity. Aims: To evaluate the pre-clinical activity of BKM120 on AML cell lines and primary samples. Methods: BKM120 (Novartis) was tested on AML cell lines (U937, NB-4, HL- 60/Mx2, OCI-AML2, HL-60, OCI-AML3, THP-1, MOLM-13 and KG-1) and on 15 primary AML samples. Given the role of PI3K/Akt/mTOR in cell metabolism, BKM120 was then combined with dichloroacetate (DCA), a glycolitic modulator, on selected AML samples. Cell cycle and apoptosis were analyzed by Acridine-Orange and AnnexinV (AnnV)/PI staining, respectively. Signaling modulations and metabolic changes were evaluated by western blot and by the XF24 Flux analyzer, respectively. In vivo experiments were performed on non-obese diabetic severe combined immunodeficient interleukin-2 receptor g-null mice. Results: Basal expression and phosphorylation levels of PI3K/Akt/mTOR pathway components were initially assessed on AML cell lines and primary samples. Despite some heterogeneity, all cell lines shared the constitutive activation of PI3K/Akt/mTOR axis. Moreover, 7 of the 9 primary samples tested showed a higher phospho/total Akt ratio than normal mononuclear cells (MNCs) and displayed p-mTOR (S2448 and S2481) and p-4EBP1 (T37/46) overexpression, suggesting the aberrant activation of PI3K/Akt/mTOR pathway. BKM120 exposure resulted in a dose-dependent dephosphorylation of Akt (S473) and Gsk3α/β (S21/9) in all cell lines tested and affected, at higher doses, mTOR activity inducing the dephosphorylation of mTOR (S2448 and S2481), p70S6K (S371) and 4EBP1 (T37/46). Blockade of PI3K/Akt/mTOR signaling inhibited cell growth (IC50s: 0.7-1.2μM) and induced a significant (p<0.005) dose- and time-dependent apoptosis in all cell lines. Cytotoxicity was preceded by a temporary G2/M block that was rapidly followed by induction of apoptosis, as demonstrated by the increase of the subG0/G1 peak at 72h. Efficacy of BKM120 was then confirmed on primary samples: at 144h, AnnV+ cells increased from 19.5±9.6% (vehicle) to 48.0±23.6% (p<0.001) at 5μM. Dephosphorylation of Akt (S473), achieved in 3/3 primary samples, supported the target inhibition. Conversely, BKM120 failed to show considerable cytotoxicity on normal and PHA-activated MNCs (8.0% and 4.4% apoptosis net increase at 5µM, respectively). Metabolic perturbations induced by BKM120 were then assessed on AML cells demonstrating a dose-dependent reduction of basal and maximal respiration as well as ATP production in both cell lines and primary samples. Furthermore, BKM120 strongly synergized (CI<0.6) with DCA to trigger apoptosis at lower doses on AML cell lines and primary samples. Finally, in vivo administration of BKM120 in a xenotransplant mouse model of AML markedly inhibited leukemia progression and induced a significant (p<0.001) improvement of overall survival. Summary and Conclusions: Our data demonstrated that BKM120, as single agent or in combination with other drugs (i.e. glycolytic modulators), has a significant anti-leukemic activity towards AML cell lines and primary samples, thus supporting its clinical evaluation as a therapeutic agent for AML patients

Denatonium as a Bitter Taste Receptor Agonist Modifies Transcriptomic Profile and Functions of Acute Myeloid Leukemia Cells.

The contribution of cell-extrinsic factors in Acute Myeloid Leukemia (AML) generation and persistence has gained interest. Bitter taste receptors (TAS2Rs) are G protein-coupled receptors known for their primary role as a central warning signal to induce aversion toward noxious or harmful substances. Nevertheless, the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. In this study, we found that AML cells express functional TAS2Rs. We also highlighted a significant association between the modulation of some TAS2Rs and the poor-prognosis AML groups, i.e., TP53- and TET2-mutated, supporting a potential role of TAS2Rs in AML cell biology. Gene expression profile analysis showed that TAS2R activation with the prototypical agonist, denatonium benzoate, significantly modulated a number of genes involved in relevant AML cellular processes. Functional assay substantiated molecular data and indicated that denatonium reduced AML cell proliferation by inducing cell cycle arrest in G0/G1 phase or induced apoptosis via caspase cascade activation. Moreover, denatonium exposure impaired AML cell motility and migratory capacity, and inhibited cellular respiration by decreasing glucose uptake and oxidative phosphorylation. In conclusion, our results in AML cells expand the observation of cancer TAS2R expression to the setting of hematological neoplasms and shed light on a role of TAS2Rs in the extrinsic regulation of leukemia cell functions

Recurrent PDL1 expression and PDL1 (CD274) copy number alterations in breast implant-associated anaplastic large-cell lymphomas

Breast implant-associated anaplastic large-cell lymphoma (BI-ALCL) is a variant of anaplastic large-cell lymphoma arising within seroma effusion associated to breast implants. BI-ALCL is a rare disease, recently recognized as a new provisional entity by the 2017 revised WHO classification. All BI-ALCLs tested so far showed a "triple negative" genetic profile - negative for ALK, DUSP22, and TP63 rearrangements - and were characterized by mutational and gene expression profiles consistent with aberrant activation of the JAK/STAT pathway. The active form of STAT3 (pSTAT3) is constantly expressed in BI-ALCLs and may favor tumor immune escape by triggering the transcription of PDL1 (CD274), a gene encoding the immune-checkpoint molecule programmed cell death ligand 1 (PDL1); immunohistochemical positivity for PDL1 has been recently described in three BI-ALCL cases, and one of them also harbored PDL1 gene amplification. We evaluated PDL1 and pSTAT expression by immunohistochemistry and PDL1 copy number alterations (CNAs) at chromosome 9p24.1 by fluorescent in situ hybridization in a cohort of nine BI-ALCL cases; we also investigated the presence of tumor infiltrating PD1+ T-cells (TILs) and PDL1+ tumor-associated macrophages (TAMs) in BI-ALCL microenvironment. Tumor cells expressed PDL1 in 5/9 cases (56%) and harbored PDL1 CNAs in 3/9 cases (33%); immunohistochemistry for pSTAT3 was positive in all 6 cases tested (100%), indicative of active JAK/STAT signaling. We observed PDL1 CNAs only among PDL1-positive cases, while PD1+ TILs and PDL1+ TAMs were present at variable levels in both PDL1-positive and PDL1-negative BI-ALCLs. We report frequent PDL1 expression and recurrent PDL1 CNAs in BI-ALCLs: our data suggest that 9p24.1 alterations represent a common mechanism of PDL1 overexpression in this disease, likely acting in synergy with constitutive pSTAT3 signaling. In PDL1-positive cases without chromosomal aberration, PDL1 expression may be induced by JAK/STAT signaling alone and/or others alternative pathways. BI-ALCL microenvironment hosts variable amounts of PD1+ TILs and PDL1+ TAMs, suggesting the presence of an active PD1/PDL1 axis. These findings may be of therapeutic value in advanced-stage patients, who may benefit from a PD1/PDL1 blocking treatment

Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells

12nononeMetformin can induce breast cancer (BC) cell apoptosis and reduce BC local and metastatic growth in preclinical models. Since Metformin is frequently used along with Aspirin or beta-blockers, we investigated the effect of Metformin, Aspirin and the beta-blocker Atenolol in several BC models. In vitro, Aspirin synergized with Metformin in inducing apoptosis of triple negative and endocrine-sensitive BC cells, and in activating AMPK in BC and in white adipose tissue (WAT) progenitors known to cooperate to BC progression. Both Aspirin and Atenolol added to the inhibitory effect of Metformin against complex I of the respiratory chain. In both immune-deficient and immune-competent preclinical models, Atenolol increased Metformin activity against angiogenesis, local and metastatic growth of HER2+ and triple negative BC. Aspirin increased the activity of Metformin only in immune-competent HER2+ BC models. Both Aspirin and Atenolol, when added to Metformin, significantly reduced the endothelial cell component of tumor vessels, whereas pericytes were reduced by the addition of Atenolol but not by the addition of Aspirin. Our data indicate that the addition of Aspirin or of Atenolol to Metformin might be beneficial for BC control, and that this activity is likely due to effects on both BC and microenvironment cells.noneTalarico, Giovanna; Orecchioni, Stefania; Dallaglio, Katiuscia; Reggiani, Francesca; Mancuso, Patrizia; Calleri, Angelica; Gregato, Giuliana; Labanca, Valentina; Rossi, Teresa; Noonan, Douglas; Albini, Adriana; Bertolini, FrancescoTalarico, Giovanna; Orecchioni, Stefania; Dallaglio, Katiuscia; Reggiani, Francesca; Mancuso, Patrizia; Calleri, Angelica; Gregato, Giuliana; Labanca, Valentina; Rossi, Teresa; Noonan, Douglas; Albini, Adriana; Bertolini, Francesc