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Common fragile sites (CFSs) are prone to chromosomal breakage and are hotspots for chromosomal rearrangements in cancer cells. We uncovered a novel function of Fanconi anemia (FA) protein FANCM in the protection of CFSs that is independent of the FA core complex and the FANCI–FANCD2 complex. FANCM, along with its binding partners FAAP24 and MHF1/2, is recruited to CFS-derived structure-prone AT-rich sequences, where it suppresses DNA double-strand break (DSB) formation and mitotic recombination in a manner dependent on FANCM translocase activity. Interestingly, we also identified an indispensable function of Rad52 in the repair of DSBs at CFS-derived AT-rich sequences, despite its nonessential function in general homologous recombination (HR) in mammalian cells. Suppression of Rad52 expression in combination with FANCM knockout drastically reduces cell and tumor growth, suggesting a synthetic lethality interaction between these two genes, which offers a potential targeted treatment strategy for FANCM-deficient tumors with Rad52 inhibition., Fanconi anemia core proteins have been linked to common fragile site stability. Here the authors shed light into the role of FANCM in common fragile site protection by suppressing double-strand break formation and mitotic recombination.

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Mechanisms that alter protein phosphatase 2A (PP2A)-dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site-directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo. Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT-I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1-dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT- or death-domain-deleted (DDD)-RIPK1, but not the kinase-domain-deleted (KDD)-RIPK1, restored FTY720-mediated necroptosis in RIPK1(-/-) MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1-/- MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34+ mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance. © 2011 by The American Society of Hematology., National Institutes of Health (NIH); US Army; NIH, National Center for Research Resources (C06 RR015455)

Immune system avoidance by cancer cells is controlled through a variety of mechanisms such as CTLA-4 and PD-1/L1, which inhibit the T-cell mediated adaptive immune response to cancer. Similar mechanisms exist for evasion from innate immunity, for example, through CD47 overexpression. On healthy cells, CD47 serves as a “don't eat me” signal by the binding to the transmembrane SIRPα protein on phagocytic cells, and prevents the engulfment of “self” by macrophages. In several cancer types, tumor cells overexpress CD47 to elude the immune system. This property of CD47 makes it an attractive target for cancer treatment. Here we describe a humanized monoclonal antibody that is a potent disruptor of the CD47/SIRPα interaction and therefore presents itself as a potential therapeutic anti-CD47 antibody. This antibody binds robustly to recombinant extracellular domains (ECD) of human and cynomolgus CD47, and to full-length human and cynomolgus CD47 expressed on HEK293 cells. Functionally, it blocks the CD47/SIRPα interaction in an ELISA-based assay with an IC50 of single-digit nM. In a cell-based assay, effect on phagocytosis by this antibody was assessed. In addition, disruption of the TSP-1/CD47 interaction has been postulated to have positive outcomes in cancer therapy and the efficacy of this antibody on inhibiting this interaction is also investigated. Finally, the effect of the antibody on Raji human Burkitt's lymphoma xenograft growth in mice will be presented. A concern with anti-CD47 therapy is the undesired targeting of red blood cells (RBCs). This antibody does not induce marked hemagglutination, suggesting a limited impact on RBCs. Taken together, our data suggest that we have developed a novel, humanized anti-CD47 antibody that can restore immune system targeting of cancer cells without inducing significant RBC agglutination. Citation Format: Joshua Oaks, Ming Wang, Hui Zou. Development of a CD47-blocking antibody as a cancer therapy [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 5623.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are aberrantly regulated at transcriptional or posttranslational levels by the constitutive kinase activity of the BCR/ABL oncoprotein. As a result, altered expression/function of RBPs leads to increased resistance to apoptotic stimuli, enhanced survival, growth advantage, and differentiation arrest of CD34+ progenitors from patients in CML blast crisis. Here, we identify the mRNAs bound to the hnRNP-A1, hnRNP-E2, hnRNP-K, and La/SSB RBPs in BCR/ABL-transformed myeloid cells. Interestingly, we found that the mRNA encoding the transcription factor E2F3 associates to hnRNP-A1 through a conserved binding site located in the E2F3 3′ untranslated region (UTR). E2F3 levels were upregulated in CML-BCCD34+ in a BCR/ABL kinase- and hnRNP-A1 shuttling-dependent manner. Moreover, by using shRNA-mediated E2F3 knock-down and BCR/ABL-transduced lineage-negative bone marrow cells from E2F3+/+ and E2F3-/- mice, we show that E2F3 expression is important for BCR/ABL clonogenic activity and in vivo leukemogenic potential. Thus, the complexity of the mRNA/RBP network, together with the discovery of E2F3 as an hnRNP-A1-regulated factor, outlines the relevant role played by RBPs in posttranscriptional regulation of CML development and progression. © 2008 by The American Society of Hematology.

During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C'3-OH) in S1P and Asp(684) in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Udgivelsesdato: 2010-Mar-5 MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL(+) leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BCCD34+ and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34(+) and CD34(+)/CD19(+) bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL) (T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.

Abstract 35 As tyrosine kinase inhibitors (TKIs) do not induce long-term response in blast crisis chronic myeloid leukemia (CML-BC) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and are unable to kill quiescent Ph+ hematopoietic stem cells, alternative therapies targeting dysregulated pathways in BCR-ABL1+ acute leukemias are needed. CRM1, a karyopherin aberrantly overexpressed in several cancers, controls the nuclear export of proteins (e.g. ABL1, SET, p53, p21, FOXO and RB) that regulate normal and malignant hematopoietic cell survival, self-renewal and proliferation. Here we show that enhanced CRM1 expression also occurs in Ph+ leukemias and the clinical stage small molecule inhibitor of CRM1, KPT-330, has a detrimental effect on malignant but not normal hematopoietic progenitors. Specifically, a substantial upregulation (≥ 65% increase) in the expression levels of CRM1 was detected by immunoblot in CD34+ progenitors from bone marrow (BM) of leukemic primary samples (n=3), when compared to CD34+ progenitors from healthy (n=3) donors. Interestingly, CRM1 protein levels in CML CD34+ cells were markedly but not totally reduced by treatment with the ABL1 kinase inhibitor Imatinib (1μM, 72h), suggesting that BCR-ABL1-driven pathways are not the only factor contributing to CRM1 upregulation. Additionally, treatment of primary CML cells with KPT-330 (1μM, 72h) resulted in a 75% reduction in BCR-ABL1 kinase activity, consistent with an interrelationship between BCR-ABL1 and CRM1 activities. Finally, the ability of BCR-ABL1 to induce CRM1 expression was demonstrated upon ectopic BCR-ABL1 expression in myeloid 32Dcl3 precursor cells in which CRM1 protein levels became ∼10-fold higher. To determine the therapeutic relevance of CRM1 inhibition, cell survival was assessed in MACS-purified CD34+ progenitors. Treatment with KPT-330 resulted in ≥80% induction of apoptosis in Ph+ (n=5) CML (2μM, 72hr) and B-ALL (500nM, 72h) CD34+ cells. Conversely, KPT-330 exerted only a minimal effect on the survival of CD34+ progenitors from healthy donor BM (15–30% Annexin V positive, KPT-330 0.5–2μM, 72h). Consistent with the existence of BCR-ABL1-independent signaling leading to increased CRM1 expression, marked apoptosis was also observed in KPT-330-treated CD34+ progenitors isolated from a Ph-negative B-ALL patient. Thus, CRM1 inhibitor-based therapies might also benefit BCR-ABL1-negative leukemias. To formally determine the effects of KPT-330 on leukemic cell survival, methylcellulose-based clonogenic assays were performed. KPT-330 treatment (1μM) resulted in almost total suppression (97% reduction) of the clonogenic potential of leukemic but not normal (30% reduction) CD34+ progenitors. Because of evidence that CRM1 directly interacts with the protein phosphatase 2A (PP2A) inhibitor SET, and that CRM1 inhibition alters trafficking of hnRNP A1, a direct regulator of the SET-PP2A interplay in Ph+ leukemias, it is conceivable that the anti-leukemic activity of KPT-330 is, at least in part, mediated by PP2A activation. Indeed, KPT-330 treatment (1μM, 48hr) of BCR-ABL1+ cell lines resulted in full restoration of PP2A activity, comparable to levels observed in BCR-ABL1-negative cells. Accordingly, KPT-330 treatment (1μM, 12h) of 32Dcl3BCR-ABL1 cells caused a nuclear accumulation (4-fold increase) and cytoplasmic decrease (95% lower) of SET protein levels, as indicated by both confocal microscopy and immunoblots of subcellular fractions. Moreover, KPT-330-treated cells showed altered hnRNP A1 cellular distribution and overall downregulation. However, unexpectedly, hnRNP A1 protein levels were proportionally higher in the cytoplasm and lower in the nucleus, suggesting that the deleterious effect of CRM1 inhibition on hnRNP A1 might not depend on direct inhibition of hnRNP-A1 nuclear export. Because KPT-330 displays favorable ADME properties and has been shown to alleviate leukemic burden using in vivo models of different cancers, we are currently testing the anti-leukemic activity of this compound in a mouse model of CML-BC. We do expect a profound inhibition of leukemogenesis, and all treated mice are currently alive with no signs of toxicity. Thus, CRM1 inhibition by KPT-330 represents a potential new therapeutic avenue which could easily be exploited in malignancies like CML-BC and Ph+ ALL that show a dismal outcome to current available therapies. Disclosures: Walker: Karyopharm Therapeutics Inc: Research Funding. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Perrotti:Karyopharm Therapeutics Inc: Research Funding.

Tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis (BC) chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Increased expression/activity of nucleocytoplasmic-shuttling heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, are critical for the expression of factors (e.g. SET/PP2A, C/EBPβ/miR-328, and c-Myc) regulating proliferation and survival of Ph+ progenitors. Because the karyopherin CRM1 controls nuclear export of hnRNPs, we assessed the therapeutic potential of CRM1 inhibitors in CML-BC and Ph+ ALL models. Thus, myeloid 32D-p210BCR-ABL1 and lymphoid Baf3-p190BCR-ABL1 progenitors were exposed to the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays revealed that KPT-185 and KPT-207 decreased cell viability by ∼80% at concentrations ranging from 150-350 nM. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70-100% viable cells) by KPT-SINE. As expected, treatment (1 μM; 48h) with these inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. As a result, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization, which is important for regulation of the PP2A inhibitor SET and, therefore, for BCR-ABL1 leukemogenesis, we assessed whether KPT-207 and KPT-185 negatively regulate PP2A activity. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3839. doi:1538-7445.AM2012-3839

Abstract 3758 Despite the remarkable response in chronic phase CML, tyrosine kinase inhibitor (TKI)-based therapies do not induce long-term response in myeloid or lymphoid blast crisis CML (CML-BC) and Ph+ ALL, and are unable to kill quiescent Ph+ hematopoietic stem cells. We reported that CML disease progression is characterized by a BCR-ABL1 dose- and kinase-dependent increase in the expression/activity of nucleocytoplasmic-shuttling of heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, E2 and K, which are essential post-transcriptional and translational modulators of critical regulators of cell proliferation, survival and differentiation of CD34+ CML-BC and/or CD34+/CD19+ Ph+ ALL progenitors including SET/PP2A, E2F3, Bcl-xL, C/EBPa, miR-328, and c-Myc. The karyopherin (a class of proteins involved in regulating nuclear import/export) chromosome region maintenance 1 (CRM1) controls the nuclear export of several hnRNPs, and because growth/survival of CML-BC and Ph+ ALL progenitors requires the aberrant cytoplasmic activity of hnRNPs A1, E2 and/or K, we have explored the therapeutic potential of CRM1 inhibitors in p210 and p190 BCR-ABL1+ cell line models of CML-BC and Ph+ ALL, respectively. Thus, the cytokine-dependent myeloid 32Dcl3 and lymphoid BaF3, and the derived cytokine-independent 32D-p210BCR-ABL1 and Baf3-p190BCR-ABL1 mouse progenitors were exposed for 48 hr to different concentrations (0–10 μM) of the CRM1 selective & potent inhibitors of nuclear export (SINE) KPT-185 and KPT-207. MTT viability assays (n=3) revealed that KPT-185 and KPT-207 decreased viability ∼80% in 32D-p210BCR-ABL1 cells (KPT-185≥207) at concentrations ranging from 150–350 nM. Similar results were obtained in Baf3-p190BCR-ABL1 cells as the EC50 was 300nM for KPT-185 and KPT-207. The KPT-SINE not only induced killing, but also affected cytokine-independent growth of BCR-ABL1+ cells: proliferation was inhibited 89% and 81% by KPT-185 and KPT-207, respectively. Notably, growth and survival of non-transformed 32Dcl3 and BaF3 cells was not affected (70–100% viable cells) at concentrations (150–350 nM) of KPT-185 and KPT-207 that impair survival of BCR-ABL1+ cells. Mechanistically, we found that KPT-SINE CRM1 inhibitors altered the nuclear/cytoplasmic ratio of hnRNPs important for BCR-ABL1 leukemogenesis. Indeed, in KPT-207-treated (1 μM; 48h) BCR-ABL1+ cells hnRNP A1, E2 and K accumulated in the cytoplasm. Likewise, KPT-185 treatment (1 μM; 48h) resulted in hnRNP A1 being sequestered in the nucleus whereas hnRNP E2 distribution was not altered and, unexpectedly, levels of hnRNP K expression were markedly decreased in both subcellular compartments. Interestingly, treatment of BCR-ABL1+ cells with KPT-185 and KPT-207 (1 μM; 48h) resulted in 75% and 50% suppression of BCR-ABL1 expression and kinase activity, respectively. Furthermore, KPT-207 also reduced Myc expression in 32D-p210BCR-ABL1 cells; this is consistent with the potential interference of KPT-207 with the proliferation/survival signals triggered by the BCR-ABL1/MAPK/hnRNP K/Myc pathway in CML-BC progenitors. Because both KPT-185 and KPT-207 significantly alter hnRNP A1 localization in addition to impairing BCR-ABL1 expression/activity, and suppression of PP2A tumor suppressor activity is essential for both p210 and p190 BCR-ABL1-driven leukemogenesis, it is highly plausible that KPT-207 and KPT-185 negatively regulate the BCR-ABL1-dependent and hnRNP A1-mediated induction of the PP2A inhibitor SET thereby rescuing PP2A phosphatase activity, which in turn decreases BCR-ABL1 expression and kinase activity, and triggers in vitro and in vivo apoptosis of primary CML-BC and Ph+ ALL progenitors. Indeed, treatment with KPT-207 and KPT-185 (250 nM; 48h) restored PP2A activity in 32D-p210BCR-ABL1 cells to levels similar to those detected in non-transformed 32Dcl3 cells. Although further investigation of KPT-207 and KPT-185 mechanism of action and assessment of their biologic/therapeutic effects in CML-BC and Ph+ ALL mouse models and primary leukemic and normal progenitors is currently ongoing, it is safe to conclude that selective nuclear export (SINE CRM1) inhibitors represent potentially powerful therapeutic tools that, if used alone or in combination with TKIs, might lead to sustained complete molecular remission in CML-BC and Ph+ ALL patients. Disclosures: Shacham: Karyopharm: Equity Ownership. Kauffman:Karyopharm: Equity Ownership.

Abstract 2494 In CD34+ bone marrow (BM) progenitors from Polycythemia Vera (PV) patients and Jak2V617F-expressing cell lines, loss of protein phosphatase 2A (PP2A) tumor suppressor activity is essential for their survival and clonogenic potential, and it depends on signals generated by the mutated Jak2 oncogenic kinase that, in turn, is also negatively regulated by PP2A. Additionally, we showed (Oaks J.J. et al., ASH 2010) that suppression of PP2A requires expression of the Jak2-regulated PP2A inhibitor SET, and it can be reverted by clinically-relevant Jak2 inhibitors or by the sphingosine analog FTY720, which reactivates PP2A and inhibits Jak2V617F expression/activity upon binding/sequestration of SET. Here we report that, unlike the effect of BCR-ABL1 in CML, SET levels do not increase in normal CD34+ BM progenitors engineered to express Jak2V617F; however, SET knock-down restores PP2A activity in both Jak2V617F cell lines and CD34+ PV progenitors, suggesting a post-translational control of SET activity. By using Jak2 and PI-3K kinase inhibitors and SET mutants, we found that PP2A inactivation in PV depends on the Jak2V617F- and PI-3Kγ-induced SET phosphorylation on serine 24. Indeed, a ∼93% restoration of PP2A activity and suppression of SET phosphorylation is achieved upon exposure of Jak2V617F+ cells to the specific PI-3Kγ (AS-604850, 1μM) inhibitor. Likewise, ∼80% loss of SET inhibitory activity is observed upon transduction of S24A but not S9A SET mutant that, albeit essential for PP2A inhibition in other cell types, behaved as wild type SET. Because both Jak2 and PI-3Kγ may regulate nitric oxide synthase (NOS2) that, in turn, inhibits PP2A activity upon induction of peroxynitrate (PN)-mediated nitration of PP2A tyrosine residues with negative regulatory function, we assessed the effect of NOS/PN modulation on the Jak2V617F-dependent inactivation of PP2A. Treatment of Jak2V617F- expressing BaF3 cells with NO donors (1 mM DPTA NONOate and 10μM SIN-1) or PN (500 μM) resulted in 2.7- and 28-fold induction of PP2A activity, respectively. Accordingly, 80% inhibition of PP2A activity was observed in parental BaF3 cells upon inhibition of NOS2 by 3-bromo-7-nitroindazole (5μM). Thus, Jak2V617F inhibits PP2A by inducing SET phosphorylation and inhibiting PP2A tyrosine nitration. Notably, co-existence of both mechanisms of Jak2V617F-dependent PP2A inactivation is also supported by the evidence that NOS2 levels increase upon shRNA-mediated downregulation of SET expression, and by the marked decrease in SET phosphorylation and increase in PP2A tyrosine nitration detected in Jak2V617F cells treated with FTY720 (2.5 mM). Interestingly, similar effects were observed upon exposure of Jak2V617F cells to non-phosphorylatable and non-immunosuppressive FTY720 derivatives (e.g. OSU-2S, QC-FTY720 and SR-FTY720) but not in cells treated with the immunosuppressive phosphorylated FTY720 (FTY720-P). In agreement with our findings indicating that the anti-leukemic activity of FTY720 in Jak2V617F+ primary progenitors and cell lines does not require the sphingosine kinase 2-dependent conversion of FTY720 into FTY720-P and, consequently, its interaction with the S1PR1 receptor (Oaks JJ. et al., ASH 2010), we have now evidence showing that a synthetic FTY720-P, unlike FTY720, impairs (81% inhibition) PP2A activity in normal hematopoietic progenitors, and that the major part of the intracellular FTY720 remains non-phosphorylated. By using S1PR1-selective agonists and S1PR1 receptor knock-down, we also found that inhibition of PP2A activity by synthetic FTY720-P is mediated by the S1PR1-dependent triggering and activation of Jak2 and PI-3Kγ. Accordingly, co-treatment of normal hematopoietic progenitor cells with FTY720-P and either the Jak2 inhibitor AG490 or the PI-3Kγ inhibitor AS-604850 antagonized the inhibitory effects of FTY720-P and restored PP2A activity to levels similar to those detected in untreated cells. Altogether our data indicate that the anti-leukemic effects of FTY720 in CD34+ PV progenitors depends on its ability to antagonize the Jak2V617F- and S1PR1-induced and PI-3Kγ-mediated inactivation of PP2A through inhibition of the PI-3Kγ-dependent SET serine 24 phosphorylation, and via binding/sequestration of phosphorylated SET that, in turn, leads to enhancement of NOS/PN-mediated PP2A activation by tyrosine nitration. Disclosures: No relevant conflicts of interest to declare.

7574 Background: Protein phosphatase 2A (PP2A) is a tumor suppressor enzyme, whose activity is inhibited by I2PP2A (inhibitor 2 PP2A) in NSCLC cell lines. Our structural, molecular, genetic, and ph...

Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assays; Western blot, confocal microscopy and FACS analyses. Results: We observed increased BCR-ABL1 expression with impaired kinase activity in quiescent CML HSCs, in which BCR-ABL1 per se is required for induction of JAK2 that subsequently activated β-catenin and inhibited PP2A. In fact, PP2A was suppressed in CML but not normal CD34+/CD38−/CD90+ HSCs. FTY720 and/or its non-immunosuppressive (S)-FTY720-OMe derivative markedly reduced survival and self-renewal of CML but not normal quiescent HSCs through BCR-ABL1 kinase-independent and PP2A-mediated JAK2 and β-catenin inhibition. Importantly, FTY720 also strongly diminished BCR-ABL1+ LT-HSC frequency in serial BM transplantation assays. Conclusions: The pharmacologic targeting of the newly-identified BCR-ABL1 kinase-independent JAK2/β-catenin interplay in quiescent HSCs with FTY720 and its derivatives, might lead to cessation of lifelong patient dependence on TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2011-LB-109

Abstract 775 We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2). Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients. To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells. Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min). Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures: Verstovsek: Incyte Corporation: Research Funding.

Abstract 1222 The molecular mechanism leading to disease progression of chronic myelogenous leukemia (CML) still remains to be identified although enhanced BCR/ABL expression and activity seems to play an important role in controlling genomic stability, differentiation, and self renewal of the leukemic cell clone undergoing blastic tranformation by affecting expression and function of RNA binding proteins (RBPs) like hnRNP A1, hnRNP E2, and hnRNP K (Perrotti D. et al. J. Clin Invest. 2010). We previously reported (Harb J. et al., ASH 2009) that a BCR-ABL1 dosage-dependence and hierarchical organization exists for the expression of hnRNP A1, hnRNP E2, and hnRNP K in cell line models of CML. In fact, as BCR/ABL levels increase, upregulated expression of hnRNP A1 is observed, followed by increased expression of hnRNP E2 and hnRNP K. HnRNP A1 and hnRNP K were also temporally expressed within Lineage- Sca-1+ c-kit+ (LSK), common myeloid progenitors (CMP), and granulocyte macrophage progenitors (GMP) in a mouse model (SCLtTA TRE-BCR/ABL) of chronic myeloid leukemia. Interestingly, hnRNP A1 and hnRNP K levels in BCR/ABL+ mouse progenitors correlated with disease severity as mice with higher levels of these RBPs presented a more progressed phenotype characterized by increased mixed lineage B220+/Mac-1+ progenitors in bone marrow and spleen when compared with mice that developed a CML-CP-like phenotype. Here we show that hnRNP A1, hnRNP E2, and hnRNP K expression levels, as well as BCR/ABL activity are different in HSC (CD34+/CD38-), CMP (CD34+/CD38+/CD45+/IL-3Ra-), and GMP (CD34+/CD38+/CD45+/IL-3Ra+) isolated from peripheral blood of patients in chronic phase (CML-CP) at diagnosis, untreated accelerated phase (CML-AP), and blast crisis (CML-BC). Interestingly, in CML-CP, the highest expression of hnRNP A1 was found in the CD34+/CD38- stem cell fraction and it gradually decreased in the more mature CMP and GMP progenitors (55% and 65% lower, respectively). By contrast, consistent with the role of hnRNP A1 as regulator of progenitor cell proliferation and survival, hnRNP A1 expression progressively increased in the HSC, CMP and GMP fractions isolated from patients in CML-AP and CML-BC. Unlike hnRNP A1, hnRNP E2 and hnRNP K were barely detected in the CD34+/CD38- from CML-CP patients but their expression was markedly pronounced in the HSC fraction of progressed CML patients. In agreement with our cell line data, expression of hnRNP A1 and hnRNP E2 in advanced CML (CML-AP and CML-BC) increases when CD34+/CD38- stem cells undergo maturation toward CMP. Conversely, it appears that hnRNP K expression is the last to increase in CML-BC, suggesting a hierarchical regulation of RBP expression during differentiation and lineage commitment. Expectedly, levels of hnRNP A1 in CMPs increase during disease progression (CP Taken together, these data further implicate RBPs hnRNP A1, hnRNP E2, and hnRNP K in CML disease progression and suggest their possible role in the control of survival of stem and primitive CML-CP progenitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 2901 FTY720 is a sphingosine analog proposed by the FDA for treating Multiple Sclerosis patients because of its immunosuppressive activity, which depends on its ability to prevent lymphocyte egress into the peripheral blood. To act as an immunosuppressive drug, FTY720 undergoes sphingosine kinase 2 (SPHK2) phosphorylation and internalization upon interaction with the sphingosine-1-phosphate receptor 1 (S1PR1). FTY720 also acts as a potent activator of protein phosphatase 2A (PP2A), a tumor suppressor found inactivated in chronic and blast crisis CML with wild type or imatinib/dasatinib-resistant BCR-ABL1, Ph+ B-ALL, KitD816V AML, Jak2V617F+ MPDs and other leukemias/lymphomas. FTY720 treatment of cell lines and primary progenitors isolated from bone marrow of patients with these malignancies, markedly suppressed leukemic cell proliferation/survival and induced apoptosis in a PP2A-dependent manner. Notably, long-term treatment with FTY720 of mice carrying these hematopoietic malignancies significantly prolonged survival and restored normal myelopoiesis without exerting any toxic effect in hematopoietic and non-hematopoietic organs. However, in vivo administration of FTY720 strongly, albeit reversibly, decreases the number of circulating B and T lymphocytes. Here we report that a synthetically phosphorylated FTY720 (FTY720-P) is unable to induce neither PP2A activation nor apoptosis of BCR-ABL1-, Jak2V617F-, or KitD816V-expressing myeloid precursors, indicating that FTY720 phosphorylation is dispensable for its anti-leukemic activity. Thus, we functionally characterize two FTY720 derivatives, QC-FTYSM and OSU-2S, which were synthesized as molecules unable to undergo SPHK2 phosphorylation. Treatment (2.5 uM; 24h) of FTY720-sensitive 32D-BCR/ABL cells with QC-FTYSM and OSU2S results in ∼80% and 40%, respectively, more efficient suppression of BCR-ABL1 expression and kinase activity than that observed with FTY720. Moreover, QC-FTYSM, OSU-2S and FTY720 (2.5uM; 0–60h) induce a progressive block of proliferation and marked induction of apoptosis of 32D-BCR/ABL cells. In fact, a 96%, 98%, and 79% decrease in viability is observed after treatment with QC-FTYSM, OSU-2S and FTY720, respectively. Notably, viability of non-transformed myeloid 32Dcl3 cells is not significantly affected by treatment with FTY720 or its derivatives. Consistent with the ability of FTY720 to induce apoptosis through rescue of PP2A activity, phosphatase assays show identical ability of FTY720, QC-FTYSM and OSU-2S to restore PP2A functionality. In fact, comparable and marked decrease in the amount of inactive Y307-phosphorylated PP2Ac was detected in 32D-BCR/ABL cells treated with FTY720 or its derivatives. To formally demonstrate that QC-FTYSM and OSU-2S lack immunosuppressive activity, we first assessed their ability to be internalized upon interaction/association with the S1PR1 receptor. Thus, cells were transduced with a GFP-tagged S1PR1 and treated with either QC-FTYSM, OSU-2S, or, as positive control, FTY720-P. Confocal microscopy revealed that treatment FTY720-P resulted in a strong S1PR1 internalization. Conversely, exposure of the cells to QC-FTYSM and OSU-2S did not alter the S1PR1 membrane localization, indicating that these molecules did not undergo SHPK2 phosphorylation. Further demonstration of the inability of these compounds to act as immunosuppressive molecules was gained upon in vivo administration of a single dose of FTY720, QC-FTYSM or OSU-2C (10 mg/kg) to wild type FVB/N mice. As expected, percentage of B220+/CD19+ circulating B-cells decreased of ∼90% in FTY720-treated animals. Conversely, the percentage of B-cells after exposure to QC-FTYSM and OSU-2S remained unchanged (≤ 1% decrease). Likewise, the number or CD4+ and CD8+ cells also was not affect by treatment with the QC-FTYSM compound. Note that effect of OSU-2S on T-cells and the toxicity profile and anti-leukemic activity of these drugs in healthy animals and mouse models of deadly leukemias (e.g. T315I+ and blast crisis CML and Ph+ ALL) as well as Ph− MPDs are currently being assessed. Altogether our data indicate that QC-FTYSM and OSU-2S represent two potentially powerful and safe drugs which could be introduced in the current therapeutic protocols for different types of hematopoietic and non-hematopoietic malignancies characterized by functional inactivation of the PP2A tumor suppressor. Disclosures: No relevant conflicts of interest to declare.

Abstract 515 The success of tyrosine kinase inhibitors as first line therapy for t(9;22) Chronic Myelogenous Leukemia (CML) depends on the addiction that Philadelphia-positive (Ph+) hematopoietic progenitors, but not quiescent Ph+ stem cells, have for BCR-ABL1 tyrosine kinase activity. We reported that the activity of the tumor suppressor PP2A is inhibited in a SET-dependent manner in CML progenitors and CD34+/CD38- stem cells from chronic phase (CP) and, to a greater extent, blast crisis (BC) CML patients (Neviani P. et al.: Cancer Cell 2005, J. Clin. Invest 2007, and ASH 2008). Restoration of PP2A activity by the immunosuppressive sphingosine analogue FTY720 markedly decreases the number of Ph+ but not normal long-term culture-initiating cells (LTC-IC) and quiescent stem cells (CFSEMAX/CD34+) by suppressing the BCR/ABL kinase-independent enhancement of b-catenin expression/transcriptional activity (Oaks JJ., et al., ASH 2009). Here we report that FTY720 induces apoptosis of Ph+ CD34+/CD38- cells independent from its phosphorylation as treatment with a phosphorylated FTY720 did not alter the number of Ph+ CFSEMAX/CD34+ cells. By contrast, two non phosphorylatable and non immunosuppressive FTY720 derivatives did significantly affect survival of Ph+ stem/progenitor cells. Interestingly, we also noted that the activity but not expression of BCR-ABL1 is considerably lower in quiescent CFSEMAX/CD34+ than CFSE+/CD34+ cells that underwent at least one division (∼80% decrease; n=3). Conversely, BCR-ABL1 expression is significantly higher in quiescent than proliferating CFSE+/CD34+ cells, suggesting that BCR-ABL1 might serve as a scaffold for other kinase(s) able to sustain survival and quiescence of Ph+ stem cells. Indeed, we found that expression of the K1172R kinase-deficient BCR-ABL1 mutant enhances expression and activity of Jak2, a kinase that is not only associated with BCR-ABL1 but is also capable of inactivating and being inactivated by PP2A. Accordingly, lentiviral shRNA-mediated BCR-ABL1 downregulation in Ph+ CD34+/CD38- stem cells resulted in marked (≥70% inhibition, P Thus, expression but not activity of the oncogenic product of the t(9;22) translocation is important for recruiting and allowing SET-mediated inhibition of PP2A and activation of Jak2; two events important for Ph+ stem cell survival and self renewal. Moreover, the ability of FTY720 and of its non-immunosuppressive derivatives to induce apoptosis of Ph+ progenitors and Ph+ but not normal quiescent stem cells emphasizes the notion that FTY720 and its derivatives represent strong and non-toxic anti-leukemic agents potentially useful not only for the treatment but, perhaps, for eradicating Ph+ leukemias. Disclosures: Holyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind to mRNA in a sequence-specific manner. We showed that hnRNP-E2 inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BC) by suppressing CEBPA mRNA translation. We report here that loss of miR-328 is induced by BCR/ABL and specifically occurs in CML-BC, and its restored expression rescues differentiation and impairs clonogenic potential of BCR/ABL+ BM progenitors. Accordingly, miR-328 increases during granulocytic differentiation of human CD34+ and mouse LSK BM stem/progenitor cells. Mechanistically, BCR/ABL uses the MAPK-hnRNP-E2 pathway to suppress C/EBPα and miR-328 expression as pharmacologic inhibition of and/or shRNAs against these molecules efficiently restore miR-328 expression. Interestingly, two functional C/EBPα binding sites are present in the miR-328 promoter and positively regulate its transcription. We also show that maturation of differentiation-arrested BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the miR-328 C-rich regions. Moreover, imatinib treatment restores miR-328 expression, thus allowing its direct binding to hnRNP E2 independent from the RISC complex. Importantly, physiological miR-328 expression decreased hnRNP E2 binding to the uORF/spacer region of endogenous CEBPA mRNA (decoy activity). This, in turn, releases CEBPA mRNA from hnRNP E2 translation inhibition and allows in vitro and in vivo BCR/ABL+ cell differentiation. Although hnRNP E2 was not found in complex with the basic RISC components in BCR/ABL+ cells, miR-328 was found associated to Dicer and Ago2, suggesting that miR-328 also acts through base-pairing with the 3′UTR of mRNA targets in a RISC-dependent manner. In fact, miR-328 suppresses PIM1 protein but not mRNA expression and this effect requires the integrity of the PIM1 3′UTR. Indeed, forced expression of a wild type, but not a kinase-deficient, PIM1 lacking the 3′UTR into miR-328-expressing cells fully rescues BCR/ABL clonogenicity, suggesting that miR-328-induced inhibition of PIM1 accounts for reduced survival of miR-328-infected CML-BCCD34+ blasts. To demonstrate that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 in the seed sequence (miR-328-Mut) while retaining its C-rich character. As expected, miR-328-Mut interacted with hnRNP-E2 and rescued C/EBPα-mediated differentiation, but did not silence PIM1 expression. Thus, the discovery of dual activities for miR-328 which affect myeloid differentiation and survival not only adds a layer to the complexity of mechanisms regulating CML-BC but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA binding proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1950.

Abstract 3259 Poster Board III-1 We have shown that the sphingosine analogue FTY720 markedly induces apoptosis of Ph(+) CML (chronic phase and blast crisis) and B-ALL progenitors regardless of their sensitivity to tyrosine kinase inhibitors through the reactivation of the tumor suppressor PP2A (protein phosphatase 2A). Here we report the identification of the molecular mechanism underlying the ability of FTY720 to activate PP2A, and its therapeutic potential towards Ph(+) and Ph(−) myeloproliferative disorders (MPDs). First, we show that the pro-apoptotic and anti-proliferative effect of FTY720 is not limited to BCR/ABL+ leukemias but can be extended to Jak2V617F-driven MPDs that are also characterized by the extensive (80% inhibition) Jak2V617F-mediated suppression of PP2A activity. In fact, shRNA-mediated interference with the hnRNP A1-SET inhibitory pathway in Jak2V617F-expressing erythro-myeloid precursors revealed that Jak2V617F, like BCR/ABL, utilizes these effectors to impair PP2A function in a dose- and kinase-dependent manner. FTY720 treatment (0.5-1mM) fully restored PP2A activity and resulted in decreased BFU-E colony formation of Jak2V617F-transduced Lin- mouse marrow progenitors. Likewise, FTY720 decreased Jak2V617F expression/activity and inhibited clonogenicity of Jak2V617F-expressing 32D-EpoR+, Ba/F3 and TF-1 cells in PP2A/SHP-1-dependent manner. Secondly, we show that PP2A is also inhibited in a SET-dependent manner in CD34+/CD38- stem cells (HSCs) from CML patients. In Ph(+) HSCs, FTY720 but not imatinib or dasatinib decreases the number of long-term culture initiating cells (LTC-IC) and quiescent stem cells (CFSEmax) through activation of BCR/ABL-independent caspase-mediated apoptosis. Importantly, enhanced self-renewal of Ph(+) HSCs seems to depend on constitutive nuclear β-catenin transcriptional activity. In fact, FTY720 or PP2Ac lentiviral transduction, but not imatinib/dasatinib, induces b-catenin inactivation/degradation as measured by in situ immunofluorescence and LET/TCF assays. The effect of FTY720 in Ph(+) HSCs relies on the PP2A ability to override the BCR/ABL-independent inactivation of the β-catenin negative regulator GSK-3β, as treatment with the GSK-3β inhibitors LiCl and SB216763 increased the CFSEmax fraction and hampered the pro-apoptotic effect of FTY720 on quiescent Ph(+) HSCs. Notably, FTY720 did not affect normal stem/progenitor cell viability. Similar results were obtained with the LSK fraction from SCL-tTA/BCR/ABL mice. Reportedly, FTY720 to act as an immunosuppressant requires phosphorylation by sphingosine kinase (SPHK) 2. To determine whether conversion of FTY720 into FTY720-P is important for its anti-leukemic activity in BCR/ABL- and Jak2V617F-expressing cells, we used growth factor-dependent hematopoietic cells transformed by the constitutive activity of these oncogenic tyrosine kinases. In Jak2V617F- and BCR/ABL-transformed cells, PP2A activation by FTY720 (2.5μM, 6h) is not changed when phosphorylation is prevented by treatment with the SPHK inhibitor dimethylsphingosine (2.5μM, 6h). Accordingly, FTY720-P did not activate PP2A and did not affect BCR/ABL- and Jak2V617F-driven colony formation, indicating that the anti-leukemic activity of FTY720 does not require its phosphorylation. Because we have shown that sphingolipid PP2A activator ceramide specifically binds to SET, thus disrupting the SET/PP2A interaction in non hematopoietic cells, we investigated the effect of BCR/ABL or Jak2 V617F and that of FTY720 on ceramide production. LS-MS mass-spectrometry showed that levels of ceramide were similar in untreated and imatinib-treated BCR/ABL+ cells. Similarly, levels of diacylglycerol (DAG) kinase-phosphorylated ceramide were unchanged by treatment with FTY720, suggesting that oncogenic tyrosine kinase-induced suppression of PP2A and FTY720-dependent PP2A reactivation are not ceramide-mediated. Accordingly, FTY720 displaces biotin-labeled C6-ceramide (10μM) from SET, as measured by ceramide affinity chromatography followed by determination of SET levels in the eluted fraction. Thus, FTY720 represents a powerful therapeutic tool as it has the potential to treat and, perhaps, eradicate Ph(+) and Ph(−) MPDs by efficiently inhibiting oncogene-dependent and -;independent signals through the reactivation of the tumor suppressor PP2A via disruption of the SET/PP2A inhibitory complex. Disclosures: Cortes: Novartis: Research Funding. Cancelas:CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Abstract 855 MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind mRNA in a sequence-specific manner. We have reported that a) hnRNP-E2 suppresses CEBPA mRNA translation and inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BCCD34+; Perrotti et al, Nat Genet 2002); and b) miR-328 expression is lost in myeloid CML-BCCD34+ progenitors (n=6) and its restored expression at physiological levels rescues granulocytic differentiation and impairs clonogenic potential of primary BCR/ABL+ blasts (Eiring et al, ASH 2007). Here we show by Northern blot, real-time PCR, and microarray analyses that miR-328 levels increase during granulocytic differentiation of normal human CD34+ and mouse Lin− BM progenitors, but not during differentiation towards erythroid, megakaryocytic or monocytic lineages. BCR/ABL uses the same MAPKERK1/2-hnRNP-E2 signaling pathway to suppress both C/EBPα and miR-328, as pharmacologic or shRNA-mediated inhibition of these molecules restored miR-328 expression in BCR/ABL+ cells. In fact, two functional C/EBPα binding sites are present in the miR-328 promoter region and C/EBPα interacts in vivo with these regulatory elements to enhance miR-328 transcription. Importantly, we also show that restored maturation of BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the C-rich regions of miR-328. Indeed, RNA-immunoprecipitation (RIP) assays demonstrated that miR-328 directly binds to hnRNP-E2 independent of the RNA-induced silencing complex (RISC). Furthermore, ectopic miR-328, but not miR-181b, resulted in decreased in vivo binding of hnRNP-E2 to the uORF/spacer region of CEBPA mRNA, thereby releasing CEBPA from hnRNP-E2 translation inhibition and rescuing C/EBPa-driven neutrophil maturation (decoy activity). Differentiation of miR-328-expressing CML-BCCD34+ blasts (88.8±2.4% post-mitotic cells) correlated with induction of C/EBPa protein expression, whereas CEBPA mRNA and hnRNP E2 protein levels remained unchanged. The existence of a direct miR-328/hnRNP-E2/CEBPA interplay was formally demonstrated in vitro using RRL-directed translation assays and in vivo using the 6.15 clone of 32D-BCR/ABL cells that do not express endogenous CEBPA mRNA and require ectopic C/EBPα (wt-uORF-CEBPA) for differentiation. Addition of miR-328, but not miR-330, to hnRNP-E2-containing RRL reactions increased newly synthesized 35S-C/EBPa levels by >100%. Likewise, forced miR-328 expression in vivo resulted in decreased hnRNP-E2 binding to CEBPA mRNA, induction of C/EBPa protein but not mRNA and rescued granulocytic differentiation of 6.15-wt-uORF-CEBPA but not vector-transduced 6.15 cells. While hnRNP-E2 was not found in complex with basic RISC components (Dicer, TRBP2 and Ago2), RIP assays detected miR-328 associated to Dicer and Ago2 in miR-328-expressing cells, suggesting that it also acts through canonical RISC-dependent base-pairing with mRNA targets. Indeed, we identified the BCR/ABL-regulated PIM1 serine-threonine kinase as a bona fide miR-328 target in BCR/ABL+ cells. Ectopic miR-328 suppressed PIM1 protein but not mRNA levels, and this effect required integrity of the miR-328 binding site present in the PIM1 3'UTR. Forced expression of a wild-type but not kinase-deficient PIM1 lacking the 3'UTR into miR-328-expressing cells fully rescued BCR/ABL clonogenicity, suggesting that miR-328-induced PIM1 suppression accounts for reduced survival of miR-328-infected BCR/ABL+ blasts. To show that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 seed sequence (miR-328-Mut) while retaining its C-rich character. Similar to wild-type miR-328, miR-328-Mut efficiently interacted with hnRNP-E2, restored C/EBPa protein expression and rescued granulocytic differentiation, but was unable to silence PIM1 in 32D-BCR/ABL cells, indicating that the C-rich character of miR-328 is essential for its decoy activity, while its seed sequence integrity is necessary for RISC-dependent pairing to mRNA targets. Thus, the discovery of dual activities for miR-328 not only adds a new layer of complexity to the mechanisms regulating CML disease progression, but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA-binding proteins. Disclosures: Cortes: Novartis: Research Funding.

Polycythemia vera (PV) is a myeloproliferative disorder (MPD) caused by a valine to phenylalanine mutation in the inhibitory JH2 “pseudokinase” domain of the signaling protein Janus Kinase 2 (Jak2). While Jak2 has several known regulators (e.g. SOCS1, SOCS3, PP2A, and SHP-1), the role played by these signaling molecules in the development of PV is still largely unclear. One of these regulators, the tumor suppressor phosphatase PP2A, has been found functionally inactivated in different hematologic myeloid and lymphoid malignancies characterized by the expression of constitutively activated oncogenic tyrosine kinases (e.g. BCR/ABL). To investigate the role of PP2A in the pathogenesis of Jak2 V617F+ MPDs and the potential therapeutic relevance of PP2A activating drugs (e.g. FTY720), we determined the effects of wild type and V617F Jak2 expression on PP2A activity and assessed the molecular and biological effects of FTY720 in hematopoietic precursor cell lines and/or primary lineage-negative bone marrow cells engineered to expressed either wild type or V617F Jak2 protein. Herein we report that PP2A activity is significantly reduced by about 82% and 78% (P< 0.01) in mJak2 V617F-transduced growth factor-dependent and erythropoietin receptor-expressing 32Dcl3 (32D-EPO) and Ba/F3 cells, respectively, compared to MigR1-transduced controls. Furthermore, addition of the PP2A activator FTY720 (2.5μM) for 10 hours restored PP2A activity to 66% and 75% respectively compared to that of MigR1 controls. Mechanistically, we demonstrated that inactivation of PP2A was due to constitutive Jak2 activity. In fact, treatment of V617F Jak2-expressing cells with Jak inhibitor I (1μM; 10 hours) restored PP2A activity to 80% of controls in 32Dcl3 cells, while 600nM was sufficient to restore activity to 108% of controls in Ba/f3 cells. Likewise, transduction of murine lineage-negative bone marrow cells with wild-type Jak2 produced a 62% reduction in PP2A activity (P

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P80% reduction in tumor burden, P

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome positive (Ph1) acute lymphoblastic leukemia (ALL) are two fatal BCR/ABL-driven leukemias against which the current therapy with Abl kinase inhibitors fails to induce a long-term response, as the majority of patients are either refractory or relapse after a few months of treatment. We recently reported that functional loss of the PP2A tumor suppressor occurs during CML disease progression and that restoration of PP2A activity impairs in vitro and in vivo BCR/ABL leukemogenesis. Here we assessed the therapeutic potential of the PP2A activator FTY720 in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. FTY720 (500 nM-2.5 mM) induces caspase-dependent apoptosis (70–98% annexin V+) and impairs the clonogenic potential (70–95% inhibition) of imatinib/dasatinib-sensitive and -resistant (T315I) p210 and p190 BCR/ABL-expressing myeloid and lymphoid progenitor cell lines (Ph1 K562, 32D-p210BCR/ABL, 32D-p210(T315I)BCR/ABL and BaF3-p190BCR/ABL), respectively, and of primary bone marrow CML-BCCD34+ (n=11) and Ph1 ALLCD34+/CD19+ (n=12) patients cells. Interestingly the cytokine (IL-3 or IL-7)-dependent growth and differentiation of normal CD34+ myeloid and CD34+/CD19+ lymphoid progenitors (n=8) is not affected by FTY720 treatment. Furthermore, pharmacologic doses of FTY720 markedly suppress leukemogenesis in SCID mice (n=13 per group) transplanted with myeloid and lymphoid progenitor cells transformed with p210BCR/ABL and p190BCR/ABL, respectively. In fact, the median survival has not yet been reached in FTY720-treated (10 mg/kg/day) BCR/ABL+ cell-injected mice. Conversely, all of untreated 32D-p210BCR/ABL, 32D-p210BCR/ABL(T315I) and BaF3-p190BCR/ABL leukemic mice died of an overt acute leukemia-like process with a median survival of 4.3, 4.8 and 4.1 weeks, respectively (P

A tight control of kinase and phosphatase activity is fundamental for normal cell growth, survival and differentiation. The deregulated kinase activity of the BCR/ABL oncoprotein is responsible for the emergence and maintenance of chronic myelogenous leukemia (CML). By contrast, PP2A, a serine-threonine phosphatase involved in the regulation of many cellular functions, was found genetically inactivated in many types of cancer. We show here that, in BCR/ABL-transformed cells and CD34+ CML blast crisis progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the physiological PP2A-inhibitor SET whose expression is enhanced by BCR/ABL and increased in blast crisis CML. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and in CD34+ CML blast crisis cells, molecular and/or pharmacological activation of PP2A leads to dephosphorylation of important regulators of proliferation and survival of CML progenitors, suppresses BCR/ABL kinase activity and promotes BCR/ABL proteasome degradation via a mechanism that requires the SHP-1 tyrosine phosphatase activity. Furthermore, PP2A activation achieved by shRNA-mediated SET knock-down or PP2Ac overexpression or treatment with the PP2A activator forskolin results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential and decreased in vivo leukemogenesis of wild type and T315I BCR/ABL-transformed myeloid cells. Thus, functional inactivation of PP2A phosphatase activity is essential for BCR/ABL leukemogenesis and, perhaps, required for transition of CML into blast crisis.