Danilo Perrotti

Role of altered growth factor receptor-mediated JAK2 signaling in growth and maintenance of human acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) is sustained by small populations of leukemia stem cells (LSCs) that can resist available treatments and represent important barriers to cure. Although previous studies have shown increased signal transducer and activator of transcription (STAT)3 and STAT5 phosphorylation in AML leukemic blasts, the role of Janus kinase (JAK) signaling in primary AML compared with normal stem cells has not been directly evaluated. We show here that JAK/STAT signaling is increased in LSCs, particularly from high-risk AML. JAK2 inhibition using small molecule inhibitors or interference RNA reduced growth of AML LSCs while sparing normal stem cells both in vitro and in vivo. Increased JAK/STAT activity was associated with increased expression and altered signaling through growth factor receptors in AML LSCs, including receptor tyrosine kinase c-KIT and FMS-related tyrosine kinase 3 (FLT3). Inhibition of c-KIT and FLT3 expression significantly inhibited JAK/STAT signaling in AML LSCs, and JAK inhibitors effectively inhibited FLT3-mutated AML LSCs. Our results indicate that JAK/STAT signaling represents an important signaling mechanism supporting AML LSC growth and survival. These studies support continued evaluation of strategies for JAK/STAT inhibition for therapeutic targeting of AML LSCs.

From the biology of PP2A to the PADs for therapy of hematologic malignancies

Over the past decades, an emerging role of phosphatases in the pathogenesis of hematologic malignancies and solid tumors has been established. The tumor-suppressor protein phosphatase 2A (PP2A) belongs to the serine–threonine phosphatases family and accounts for the majority of serine–threonine phosphatase activity in eukaryotic cells. Numerous studies have shown that inhibition of PP2A expression and/or function may contribute to leukemogenesis in several hematological malignancies. Likewise, overexpression or aberrant expression of physiologic PP2A inhibitory molecules (e.g., SET and its associated SETBP1 and CIP2A) may turn off PP2A function and participate to leukemic progression. The discovery of PP2A as tumor suppressor has prompted the evaluation of the safety and the efficacy of new compounds, which can restore PP2A activity in leukemic cells. Although further studies are needed to better understand how PP2A acts in the intricate phosphatases/kinases cancer network, the results reviewed herein strongly support the development on new PP2A-activating drugs and the immediate introduction of those available into clinical protocols for leukemia patients refractory or resistant to current available therapies.

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.

Precision immunotherapy, mutational landscape, and emerging tools to optimize clinical outcomes in patients with classical myeloproliferative neoplasms

Following the 47th American Society of Hematology Meeting in 2005, the late John Goldman and Tariq Mughal commenced a conference, the 1st Post‐ASH Workshop, which brought together clinicians and scientists, to accelerate the adoption of new therapies for patients with myeloproliferative neoplasms (MPNs). The concept began with recognition of the CML success story following imatinib therapy, the discovery of JAK2V617F, and the demonstration that BCR‐ABL1‐negative MPNs are driven by abnormal JAK2 activation. This review is based on the presentations and deliberations at the XIIth Post‐ASH Workshop on BCR‐ABL1 positive and negative MPNs that took place on December 12 to 13, 2017, in Atlanta, Georgia, immediately following the 59th American Society of Hematology Meeting. We have selected some of the translational research and clinical topics, rather than an account of the proceedings. We discuss the role of immunotherapy in MPNs and the impact of the mutational landscape on TKI treatment in CML. We also consider how we might reduce TKI cardiovascular side effects, the potential role of nutrition as adjunctive nonpharmacologic intervention to reduce chronic inflammation in MPNs, and novel investigational therapies for MPNs.

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.

A "RANning" leap with "XPOrt" into TKI resistance.

In this issue of Blood , Khorashad et al[1][1] show that genetic (eg, short hairpin RNA [shRNA]-mediated) or pharmacologic (eg, KPT-330 [selinexor]) inhibition of nucleocytoplasmic protein trafficking restored sensitivity to tyrosine kinase inhibitors (TKIs) and impaired clonogenic potential of