Emilia Białopiotrowicz

FOXO1 activation is an effector of SYK and AKT inhibition in tonic BCR signal-dependent diffuse large B-cell lymphomas.

Inhibition of spleen tyrosine kinase (SYK) in tonic B-cell receptor (BCR) signal-dependent diffuse large B-cell lymphomas (DLBCLs) inhibits cellular proliferation, decreases cholesterol biosynthesis, and triggers apoptosis, at least in part via a mechanism involving decreased activity of phosphatidylinositol 3-kinase/AKT axis. Because forkhead box O1 (FOXO1) is a major effector of this pathway, we investigated the role of FOXO1 in toxicity of BCR pathway inhibition. Inhibition of SYK in DLBCL cells with tonic BCR signaling decreased phospho-AKT and phospho-FOXO1 levels and triggered FOXO1-driven gene expression. Introduction of constitutively active FOXO1 mutant triggered cell cycle arrest and apoptosis, indicating that increased FOXO1 activity is toxic to these DLBCL cells. Depletion of FOXO1 with short hairpin RNA led to almost complete resistance to chemical SYK inhibitor R406, demonstrating that FOXO1 is also required for R406-induced cell death. FOXO1 in these cells is also involved in regulation of expression of the critical master regulator of cholesterol biosynthesis, SREBP1. Because HRK is the key effector of SYK inhibition, we characterized a mechanism linking FOXO1 activation and HRK induction that involves caspase-dependent cleavage of HRKs transcriptional repressor DREAM. Because AKT in lymphoma cells can be regulated by other signals than BCR, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic FOXO1-dependent toxicity. In primary DLBCLs, FOXO1 expression was present in 80% of tumors, correlated with SYK activity, and was associated with longer overall survival. These results demonstrate that FOXO1 is required for SYK and AKT inhibitor-induced toxicity.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1–TXN–p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy.

Resistance to glucocorticosteroids (GCs) is a major adverse prognostic factor in B-ALL, but the molecular mechanisms leading to GC resistance are not completely understood. Herein, we sought to elucidate the molecular background of GC resistance in B-ALL and characterize the therapeutic potential of targeted intervention in these mechanisms. Using exploratory bioinformatic approaches, we found that resistant cells exhibited significantly higher expression of MEK/ERK (MAPK) pathway components. We found that GC-resistant ALL cell lines had markedly higher baseline activity of MEK and small-molecule MEK1/2 inhibitor selumetinib increased GCs-induced cell death. MEK inhibitor similarly increased in vitro dexamethasone activity in primary ALL blasts from 19 of 22 tested patients. To further confirm these observations, we overexpressed a constitutively active MEK mutant in GC-sensitive cells and found that forced MEK activity induced resistance to dexamethasone. Since recent studies highlight the role GC-induced autophagy upstream of apoptotic cell death, we assessed LC3 processing, MDC staining and GFP-LC3 relocalization in cells incubated with either DEX, SEL or combination of drugs. Unlike either drug alone, only their combination markedly increased these markers of autophagy. These changes were associated with decreased mTOR activity and blocked 4E-BP1 phosphorylation. In cells with silenced beclin-1 (BCN1), required for autophagosome formation, the synergy of DEX and SEL was markedly reduced. Taken together, we show that MEK inhibitor selumetinib enhances dexamethasone toxicity in GC-resistant B-ALL cells. The underlying mechanism of this interaction involves inhibition of mTOR signaling pathway and modulation of autophagy markers, likely reflecting induction of this process and required for cell death. Thus, our data demonstrate that modulation of MEK/ERK pathway is an attractive therapeutic strategy overcoming GC resistance in B-ALL patients.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Expression of PIM kinases in Reed-Sternberg cells fosters immune privilege and tumor cell survival in Hodgkin lymphoma

Reed-Sternberg (RS) cells of classical Hodgkin lymphoma (cHL) express multiple immunoregulatory proteins that shape the cHL microenvironment and allow tumor cells to evade immune surveillance. Expression of certain immunoregulatory proteins is modulated by prosurvival transcription factors, such as NFκB and STATs. Because these factors also induce expression of the oncogenic PIM1/2/3 serine/threonine kinases, and as PIMs modulate transcriptional activity of NFκB and STATs, we hypothesized that these kinases support RS cell survival and foster their immune privilege. Here, we investigated PIM1/2/3 expression in cHL and assessed their role in developing RS cell immune privilege and survival. PIM1/2/3 were ubiquitously expressed in primary and cultured RS cells, and their expression was driven by JAK-STAT and NFκB activity. Genetic or chemical PIM inhibition with a newly developed pan-PIM inhibitor, SEL24-B489, induced RS cell apoptosis. PIM inhibition decreased cap-dependent protein translation, blocked JAK-STAT signaling, and markedly attenuated NFκB-dependent gene expression. In a cHL xenograft model, SEL24-B489 delayed tumor growth by 95.8% (P = .0002). Furthermore, SEL24-B489 decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. In coculture experiments, T cells incubated with SEL24-B489-treated RS cells exhibited higher expression of activation markers than T cells coincubated with control RS cells. Taken together, our data indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link these cells with their niches. Thus, PIM kinases are promising therapeutic targets in cHL.

Abstract 1749: Preclinical characterization of SEL24-B489, a dual PIM/FLT3 inhibitor for the treatment of hematological malignancies

PIM kinases represent an emerging therapeutic target in multiple hematological malignancies, as exemplified by currently ongoing phase I clinical trials by Astra Zeneca (AZD1208) and Novartis (LGH447) in acute myeloid leukemia and multiple myeloma. Selvita has developed a potent and selective dual PIM/FLT3 mutant kinase inhibitor - SEL24-B489 showing high inhibitory activity on all three PIM kinase isoforms and FLT3 kinase mutants. We have previously reported that PIM kinases are important downstream effectors of FLT3 signaling and play a crucial role in cell survival and inhibition of apoptosis upon expression. Due to heterogeneous nature of AML, dual inhibition of FLT3 mutant kinase and PIM kinases led to improved efficacy of our compound in comparison to selective inhibitors of either PIM of FLT3 kinases. Herewith, we would like to report further progress of characterizing the B489 inhibitor beyond AML. We assessed PIM kinase expression levels in a panel of lymphoid malignancies and found that PIM1 and PIM2 exhibit high expression levels in a fraction of mantle cell lymphoma (MCL), diffuse large-B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin9s lymphoma (HL), chronic lymphocytic leukemia (CLL) and mucosa associated lymphoid tissue-type (MALT) lymphoma cell lines and primary tumors . High levels of PIM kinases were associated with certain established adverse prognostic factors and clinical outcome of the patients and correlated with aggressiveness of the disease in some of these tumors. Inhibition of PIM kinases with tool inhibitors was shown to influence cellular proliferation and, translational inhibition of 4EBP1 as reported in the literature. In addition, SEL24-B489 inhibited NFκB activity and decreased CXCR4 expression. Comparison of SEL24-B489 to competitive PIM inhibitors revealed higher cellular activity and biomarker response, as shown by inhibition of phospho-S6 phosphorylation in sub-microM concentrations. The presented data will further validate SEL24-B489 as a successful example of rational drug design and present a promising therapeutic approach in multiple hematological malignancies, both stand alone and in combination with standard of care and targeted therapies in clinical development. Citation Format: Wojciech Czardybon, Renata Windak, Izabela Dolata, Magdalena Salwinska, Maciej Szydlowski, Tomasz Sewastianik, Emilia Bialopiotrowicz, Elzbieta Mądro, Ewa Lech-Maranda, Bozena K. Budziszewska, Katarzyna Borg, Przemyslaw Juszczynski, Krzysztof D. Brzozka. Preclinical characterization of SEL24-B489, a dual PIM/FLT3 inhibitor for the treatment of hematological malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1749. doi:10.1158/1538-7445.AM2014-1749

Microenvironment‐induced PIM kinases promote CXCR4‐triggered mTOR pathway required for chronic lymphocytic leukaemia cell migration

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment‐dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first‐line treatment. In primary CLL cells, inhibition of PIM kinases with a pan‐PIM inhibitor, SEL24‐B489, decreased PIM‐specific substrate phosphorylation and induced dose‐dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24‐B489 was similar in TP53‐mutant and TP53 wild‐type cells. Finally, inhibition of PIM kinases decreased CXCR4‐mediated cell chemotaxis in two related mechanisms‐by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4‐triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12‐triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment‐modulated PIM expression, their pro‐survival function and a role of PIMs in CXCR4‐induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.

Abstract 5394: First-in-class dual PIM/FLT3 kinase inhibitor SEL24-B489 for the treatment of hematological malignancies

Despite huge effort spent on understanding acute myeloid leukemia (AML), current standards of care are still based on the same chemotherapy agents as two decades ago - typically based on cytarabine chemotherapy with an anthracycline. Although patients aged PIM kinases are expressed in various cancers including AML but also other liquid tumors as well as in some solid tumors. Inhibition of PIM kinases which are downstream in the FLT3 signaling cascade have already shown influence on cancer cell survival. In addition there are great hopes related to the fact that PIM kinases were shown to contribute in resistance to FLT3 inhibitors. This leads to the conclusion that combined inhibition of PIM and FLT3 may be a rational strategy. Selvita has developed series of inhibitors combining activity against PIM and FLT3 kinases. Selected clinical candidate SEL24-B489 - has shown excellent anticancer efficacy. It is active against broad panel of AML cell lines and primary blasts, but also against other hematological cancers: DLBCL, CLL, HL - both in vitro on cell lines, patient samples and in vivo. Head to head comparison of SEL24-B489 with PIM (AZD1208) and FLT3 (AC220) inhibitors currently in clinical development will be presented. Comparison shows strong activity of SEL24-B489 against broader panel of cell lines in vitro and in vivo (in xenograft models) than AZD1208 or AC220. SEL24-B489 is currently in preclinical development and details of toxicology profile will also be discussed. Citation Format: Krzysztof Brzozka, Wojciech Czardybon, Aniela Golas, Renata Windak, Michal Galezowski, Ewelina Gabor-Worwa, Bozena Winnik, Agnieszka Przybylowicz, Maciej Szydlowski, Emilia Bialopiotrowicz, Tomasz Sewastianik, Elzbieta Mądro, Ewa Lech-Maranda, Krzysztof Warzocha, Przemyslaw Juszczynski. First-in-class dual PIM/FLT3 kinase inhibitor SEL24-B489 for the treatment of hematological malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5394. doi:10.1158/1538-7445.AM2015-5394