Giovannino Silvestri

Rapid recognition of drug-resistance/sensitivity in leukemic cells by Fourier transform infrared microspectroscopy and unsupervised hierarchical cluster analysis

We tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in leukemic cells exposed to tyrosine kinase inhibitors (TKIs). Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). Mouse-derived pro-B Ba/F3 cells transfected with and stably expressing the human p210(BCR-ABL) drug-sensitive wild-type BCR-ABL or the V299L or T315I p210(BCR-ABL) drug-resistant BCR-ABL mutants were exposed to imatinib-mesylate (IMA) or dasatinib (DAS). MicroFTIR was carried out at the Diamond IR beamline MIRIAM where the mid-IR absorbance spectra of individual Ba/F3 cells were acquired using the high brilliance IR synchrotron radiation (SR) via aperture of 15 × 15 μm(2) in sizes. A conventional IR source (globar) was used to compare average spectra over 15 cells or more. IR signatures of drug actions were identified by supervised analyses in the spectra of TKI-sensitive cells. Unsupervised HCA applied to selected intervals of wavenumber allowed us to classify the IR patterns of viable (drug-resistant) and apoptotic (drug-sensitive) cells with an accuracy of >95%. The results from microFTIR + HCA analysis were cross-validated with those obtained via immunochemical methods, i.e. immunoblotting and flow cytometry (FC) that resulted directly and significantly correlated. We conclude that this combined microFTIR + HCA method potentially represents a rapid, convenient and robust screening approach to study the impact of drugs in leukemic cells as well as in peripheral blasts from patients in clinical trials with new anti-leukemic drugs.

Cellular and Molecular Networks in Chronic Myeloid Leukemia: the leukemic stem, progenitor and stromal cell interplay.

The use of imatinib, second and third generation ABL tyrosine kinase inhibitors (TKI) (i.e. dasatinib, nilotinib, bosutinib and ponatinib) made CML a clinically manageable and, in a small percentage of cases, a cured disease. TKI therapy also turned CML blastic transformation into a rare event; however, disease progression still occurs in those patients who are refractory, not compliant with TKI therapy or develop resistance to multiple TKIs. In the past few years, it became clear that the BCRABL1 oncogene does not operate alone to drive disease emergence, maintenance and progression. Indeed, it seems that bone marrow (BM) microenvironment-generated signals and cell autonomous BCRABL1 kinase-independent genetic and epigenetic alterations all contribute to: i. persistence of a quiescent leukemic stem cell (LSC) reservoir, ii. innate or acquired resistance to TKIs, and iii. progression into the fatal blast crisis stage. Herein, we review the intricate leukemic network in which aberrant, but finely tuned, survival, mitogenic and self-renewal signals are generated by leukemic progenitors, stromal cells, immune cells and metabolic microenvironmental conditions (e.g. hypoxia) to promote LSC maintenance and blastic transformation.

Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia

The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation.

BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia

The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the −11.7 kb and −0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34− cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.

Abstract 4736: Bone marrow microenvironment-induced miR-300 expression impairs natural killer cell proliferation and antitumor activity

Natural killer (NK) cells mediate immune responses against cancer; however, NK cell quantitative and functional defects are features of cancers including chronic myelogenous leukemia (CML). As increased numbers of activated NK cells were found in CML patients in treatment-free remission, the understanding of the molecular events inhibiting NK proliferation and function may lead to the development of NK cell-based therapies against drug-resistant cancer stem cells. Because altered miRNA expression and inactivation of the protein phosphatase 2A (PP2A) tumor suppressor are also features of cancer, and SET-dependent PP2A inhibition is essential for NK cell function, we hypothesized that increased expression of miR-300, a miRNA with antiproliferative activity, found inhibited in CML and targeting the PP2A inhibitor SET, accounts for impaired NK cell proliferation/activity. An initial analysis revealed that miR-300 levels were significantly higher in peripheral blood (PB) CD56 + CD3 - NK cells from CML patients at diagnosis compared to healthy individuals. As NK cell activity is regulated by the bone marrow microenvironment (BMM), we evaluated whether hypoxic conditions and/or cell-to-cell interaction influence NK cell proliferation and cytotoxic activity by modulating miR-300 intracellular levels. A marked and significant increase in miR-300 expression was detected in NK-92 and primary CD56 + CD3 - NK cells exposed to low O 2 levels or cultured in the presence of conditioned medium (CM) or exosomes isolated from BM-derived primary mesenchymal stromal (MSC) and HS-5 MSC cells. As expected, increased miR-300 levels correlated with decreased SET levels and markedly reduced NK cell number. Interestingly, miR-300-induced growth inhibition was rescued in NK cells treated with CM/exosomes from HS-5 cells expressing an anti-miR-300 lentivirus. Notably, qRT-PCR indicated that miR-300 was contained in MSC exosomes. Functionally, exposure to MSCs (CM or exosomes) inhibited NK cell IL-12/IL-18-induced IFN-γ production and cytotoxic activity against K562 CML-BC cells in a miR-300-dependent manner. Accordingly, miR-300 lentiviruses and/or CpG-miR-300 oligonucleotides inhibited SET expression, reduced proliferation and suppressed spontaneous cytotoxicity of NK-92 and/or primary NK cells, likely through reactivation of PP2A. Because BM hypoxic conditions and MSCs significantly contribute to decreased NK cell number and cytotoxic activity through upregulation of miR-300, its genetic or pharmacologic inhibition may result in reactivation of NK cell activity against leukemic stem/progenitor cells. This work is supported in part by NIH-NCI RO1CA163800. Citation Format: Rossana Trotta, Giovannino Silvestri, Lorenzo Stramucci, Martin Guimond, Guido Marcucci, Xiaoxuan Fan, Maria R. Baer, Danilo Perrotti. Bone marrow microenvironment-induced miR-300 expression impairs natural killer cell proliferation and antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4736.