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Supplementary Figures S1-S4. Suppplementary S1: Western Blot analysis o study the effect of the combinatory therapy on DDR Supplementary S2: Evaluation of the percentage of hyper damaged cells among the leukemic compartment Supplementary S3: Cell cycle analysis on OCI-AML2 treated cells Supplementary S4: Model of the mechanism of action of the combinatory therapy PARPi and 5FU

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA. Mol Cancer Ther; 14(4); 889–98. ©2015 AACR.

Genetic and morphological characteristics of AML patients and healthy donors used for miR-181a/b expression and RASSF1A protein analysis.

Western blot analysis of p53 protein in puromycin and gfp sorted miR-ZIP-expressing NB4 cells. GAPDH was used as internal control. Values below the blots represent the median {plus minus} SD from three independent experiments.

Primer sequences used for mRNA applification by qPCR.

Genetic and morphological characteristics of APL patients used for miR-181a/b expression analysis.

A, Apoptosis assay in NB4 cells 24h and 48h after application of As2O3 (1 µM). B, FACS analysis for CD11b expression in As2O3 treated NB4 cells 48h after application. C, qPCR analysis for C/EBPβ mRNA expression in NB4 cells after As2O3 application. D, Apoptosis assay in NB4 cells 48h after application of doxorubicin (0.01 µg/ml) with or without cytarabine (0.05 µM). E, qPCR analys for C/EBPβ mRNA expression in NB4 cells after doxorubicin and cytarabine application. For qPCR measurement GAPDH was used as control. Bars and values in the dot blots represent the median {plus minus} SD from three independent experiments.* p-value {less than or equal to} 0.05; ** p-value {less than or equal to} 0.01.

Background Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. Methods The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. Results We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. Conclusions Our data suggest that WEE1 plays a role in ALL blast’s survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL. Electronic supplementary material The online version of this article (10.1186/s13045-018-0641-1) contains supplementary material, which is available to authorized users.

The existing treatments to cure acute leukemias seem to be nonspecific and suboptimal for most patients, drawing attention to the need of new therapeutic strategies. In the last decade the anticancer potential of poly ADP-ribose polymerase (PARP) inhibitors became apparent and now several PARP inhibitors are being developed to treat various malignancies. So far, the usage of PARP inhibitors has been mainly focused on the treatment of solid tumors and not too much about their efficacy on leukemias is known. In this study we test, for the first time on leukemic cells, a combined therapy that associates the conventional chemotherapeutic agent fluorouracil (5FU), used as a source of DNA damage, and a PARP inhibitor, rucaparib. We demonstrate the efficacy and the specificity of this combined therapy in killing both acute myeloid leukemia and acute lymphoid leukemia cells in vitro and in vivo. We clearly show that the inhibition of DNA repair induced by rucaparib is synthetic lethal with the DNA damage caused by 5FU in leukemic cells. Therefore, we propose a new therapeutic strategy able to enhance the cytotoxic effect of DNA-damaging agents in leukemia cells via inhibiting the repair of damaged DNA. Mol Cancer Ther; 14(4); 889–98. ©2015 AACR.

Since their appearance, humanized mice carrying human immune system seemed promising tools to study the crosstalk between cancer and immunity. The NOD-scidIL2Rgammanull (NSG) mice engrafted with human cord blood (hCB)-CD34+ cells have been proposed to be a valuable tool to reproduce human immune system in mouse. However, the lack of solid evidences on the functionality of their human immune components limits their usage in immune-oncology. We report that (hCB)-CD34+ cells lose their ability to propagate and originate bone marrow-derived human immune cells after two serial transplantations in NSG mice. We demonstrate that transplants of bone marrow patient-derived acute myeloid leukemias (hAMLs) grow very similarly in the humanized (hCB)-CD34+ NSG and parental NSG mice. The similar extent of engraftment and development of leukemias in (hCB)-CD34+ NSG and controls suggests a poor human immune response against not compatible hAMLs. Our findings suggest that (hCB)-CD34+ NSG mice are transient and/or incomplete carriers of the human immune system and, therefore, represent a suboptimal tool to study the interaction between tumor and immune cells.

Although improvements in knowledge of genetic basis of ALL have been made and many new drugs are now available, the prognosis for adult patients with ALL is still a challenge. Current therapies based on chemotherapy regimens or on tailored targets do not completely eradicate the leukemia clone and relapse is a predictable event. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), an ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Chk1 and Chk2 are serine/threonine kinases which following activation by a broad spectrum of damaged DNA coordinate the DNA damage response. By quantitative PCR, higher transcript levels of Chk1 were found in 8 leukemia cell lines and 54 newly diagnosed ALL cases compared to normal bone marrow mononuclear cells (p value < 0.001). Immunohistochemistry of formalin-fixed paraffin-embedded tissue samples collected from 60 ALL patients showed a diffuse positivity for Chk1, Chk2 and the phosphorylated forms of Chk1 (Ser345) in 51/54 (96%), 55/57 (94%) and 45/56 (80%) of the cases respectively, whereas 15/55 (27%) of ALLs stained for phosphorylated form of Chk2 (Thr68). Interestingly, in our ALL series, genomic damage was suggested by the nuclear labeling for γ-H2A.X molecule in 40/59 (68%) of samples. In B-/T-ALL cell lines (BV-173, SUP-B15, REH, NALM-6, NALM-19, MOLT-4, RPMI8402 and CCRF-CEM) inhibition of Chk1 resulted in a dose and time-dependent cytotoxicity (IC50 from 57.4 nM to 1423.0 at 24 hours), induction of apoptosis and increased DNA damage as demonstrated by increased levels of phosphorylated Chk1 (Ser317 and Ser345) and H2A.X (Ser139). Moreover, PF-00477736 efficiently triggered the Chk1-Cdc25-Cdc2 pathway with a decreased phosphorylation of Cdc25c (Ser216) and Cdc2 (Tyr15). Gene expression profiling analysis shed light on molecular mechanisms driven by PF-00477736 revealing a deregulation of genes involved in DNA damage checkpoint, cell cycle control and apoptosis. Among these, Gadd45a and Plk3 were confirmed by qPCR analysis up-regulated, whereas Cdk4 and Chk2 resulted down-regulated between treated and untreated samples. The efficacy of PF-00477736 as a single agent was confirmed ex-vivo in primary leukemic blasts separated from 14 ALL patients but not in in primary cultures of normal bone marrow mononuclear cells. Furthermore, in mice transplanted with T-lymphoid leukemia PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). In conclusion, in vitro, ex-vivo and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia and they provide a strong rationale for its future clinical investigation.

Mutations can confer a selective advantage on specific cells, enabling them to go through the multistep process that leads to malignant transformation. The cancer stem cell hypothesis postulates that only a small pool of low-cycling stem-like cells is necessary and sufficient to originate and develop the disease. Normal and cancer stem cells share important functional similarities such as ‘self-renewal’ and differentiation potential. However, normal and cancer stem cells have different biological behaviours, mainly because of a profound deregulation of self-renewal capability in cancer stem cells. Differences in mode of division, cell-cycle properties, replicative potential and handling of DNA damage, in addition to the activation/inactivation of cancer-specific molecular pathways confer on cancer stem cells a malignant phenotype. In the last decade, much effort has been devoted to unravel the complex dynamics underlying cancer stem cell-specific characteristics. However, further studies are required to identify cancer stem cell-specific markers and targets that can help to confirm the cancer stem cell hypothesis and develop novel cancer stem cell-based therapeutic approaches.

Background/Aims Hepcidin is a liver-produced hormone that regulates systemic iron homeostasis. Hepcidin expression is stimulated upon iron overload or inflammation while iron deficiency, anemia and tissue hypoxia are negative regulators. We investigated the involvement of 2-oxoglutarate-dependent oxygenases, HIF-1 and other transcription factors in the hypoxic suppression of hepcidin. Methods Northern blotting analysis and real time PCR were used to determine hepcidin mRNA levels in hepatoma cells and hepcidin promoter activity was measured using Huh7 cells transfected with suitable reporter constructs under various conditions. Results Treatment of human cultured hepatoma cells with hypoxia or known inhibitors of 2-oxoglutarate-dependent oxygenases, such as the iron chelator desferrioxamine, cobalt or the 2-oxoglutarate analogue dimethyl-oxalylglycine significantly reduced hepcidin mRNA levels and down-regulated its gene promoter activity. This effect was not dependent on the HREs or other known putative response elements in the hepcidin promoter and was observed even under interleukin-6 treatment. Conclusions 2-Oxoglutarate-dependent oxygenases are important to maintain high hepcidin mRNA expression in a HIF-1-independent manner. We suggest that modulation of oxygenase activity may be of therapeutic value in iron-related disorders.

Background Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Methods The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent n-ethyl-n-nitrosourea. Results Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia. Conclusions In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0206-5) contains supplementary material, which is available to authorized users.

In acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. microRNAs are known to be critical players in the formation of the leukemic phenotype. In this study, we report downregulation of the miR-181a/b gene cluster in APL blasts and NB4 leukemia cells upon ATRA treatment as a key event in the drug response. We found that miR-181a/b expression was activated by the PML/RARα oncogene in cells and transgenic knock-in mice, an observation confirmed and extended by evidence of enhanced expression of miR-181a/b in APL patient specimens. RNA interference (RNAi)-mediated attenuation of miR-181a/b expression in NB4 cells was sufficient to reduce colony-forming capacity, proliferation, and survival. Mechanistic investigations revealed that miR-181a/b targets the ATRA-regulated tumor suppressor gene RASSF1A by direct binding to its 3′-untranslated region. Enforced expression of miR-181a/b or RNAi-mediated attenuation of RASSF1A inhibited ATRA-induced granulocytic differentiation via regulation of the cell-cycle regulator cyclin D1. Conversely, RASSF1A overexpression enhanced apoptosis. Finally, RASSF1A levels were reduced in PML/RARα knock-in mice and APL patient samples. Taken together, our results define miR-181a and miR-181b as oncomiRs in PML/RARα-associated APL, and they reveal RASSF1A as a pivotal element in the granulocytic differentiation program induced by ATRA in APL. Cancer Res; 75(16); 3411–24. ©2015 AACR.

Hepcidin is a beta-defensin-like peptide and a principle regulator of systemic iron homeostasis. In concordance with this dual function its expression is modulated by systemic iron requirements and in response to infectious and inflammatory stimuli. Studies of hepcidin provide novel insight into the molecular mechanisms involved in maintaining iron homeostasis in the healthy state and iron redistribution in response to chronic infections and inflammation. Furthermore, a deregulation of hepcidin may cause elevated intestinal iron absorption that hallmarks a group of frequent iron overload disorders, the Hereditary Hemochromatosis. The aim of this review is to discuss hepcidin function in iron-homeostasis under normal physiological and pathophysiological conditions.

The peptide and metallocene-peptide bioconjugates R-Arg-Arg-Phe-NH2, R-Phe-Arg-Phe-NH2 where R = H, Fe(Cp)(C5H4–CO), Co(Cp)(C5H4–CO)+ and R′-Gly-Trp-Arg-Arg-Phe-NH2, R′-Trp-Arg-Arg-Phe-NH2, where R′ = n-C5H11CO, Fe(Cp)(C5H4–CO), Co(Cp)(C5H4–CO)+, and Arg = l -arginine, Gly = l -glycine, Phe = l -phenylalanine, Trp = l -tryptophan were prepared by solid phase peptide synthesis (SPPS). The compounds were purified by RP-HPLC and characterized by ESI–MS and NMR spectroscopy. Antibacterial properties of the compounds were determined by minimum inhibitory concentration (MIC) tests against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.

Host defense peptides (HDPs) are endogenous antibiotics that play a multifunctional role in the innate immunity of mammals. Among these, βdefensins contribute to mucosal and epithelial defense, also acting as signal molecules for cellular components of innate and adaptive immunity. Numerous members of this family have been identified in mammalian and avian species, and genomic studies in human and mouse indicate a considerable complexity in their gene organization. Recent reports on the evolution of primate and rodent members of this family indicate quite a complex pattern of variation. In this review we briefly discuss the evolution of mammalian βdefensins in relation to other types of defensins, and then concentrate on the evolution of βdefensins 1 to 4 in primates. In particular, the surprisingly varied patterns of evolution, which range from neutral or weakly purifying, to positive selection to a high level of conservation are analyzed in terms of possible genetics, structural or functional implications, as well as to observed variations on the antimicrobial activity in vitro. The role of polymorphisms in the genes encoding for these host defense peptides in determining susceptibility to human diseases are also briefly considered.

Abstract 76 Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases regulated by Ataxia-Telangiectasia and Rad3-related (ATR) kinases and involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Deregulation of these pathways has been previously described in BCR-ABL positive cells and involved in chemoresistance. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, in this study we aimed to investigate the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in Ph+ ALL and to determine potential biomarkers of functional inhibition. We first examined Chk1 and Chk2 mRNA expression levels in 45 newly diagnosed Ph+ ALL patients, in their paired remission samples, in 14 relapsed cases and in 3 Ph+ cell lines (BV-173, SUPB-15 and K562) by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation). Higher transcript levels of Chk1 but not Chk2 were found in newly diagnosed patients compared to remission samples (p = 0.0009 for Chk1 and p= 0.8183 for Chk2). Chk1 transcript levels were comparable between diagnosis and relapsed cases (p = 0.5728), suggesting that the ATR-Chk1 pathway is strongly activated in Bcr-Abl-positive cells. This was confirmed by phosphorylation of Chk1 Ser317 by western blotting analysis in Ph+ cell lines. We then evaluated the effect of PF-0477736 as single agent on cell viability using the Cell Proliferation Reagent WST-1 (Roche). BV-173, SUPB-15 and K562 cell lines were incubated with increasing concentration of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1–0.5 μM, with BV-173 being the most sensitive, while K562 the most resistant. These results were confirmed in primary blasts cells from a Ph+ ALL patient with wild-type Bcr-Abl and from 3 cases harboring the T315I Bcr-Abl mutation found to be insensitive to the available TKIs (IC50 ranged from 0.1–0.5 μM at 48 hours). Consistent with the WST-1 results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in both cell lines and primary cells. To test whether increased apoptosis resulted from Chk1 inhibition, we assessed the changes in phosphorylation of Cdc25c phosphatase, which is inactivated by Chk1 to prevent mitotic entry, and of γH2AX, which is increased in response to DNA damage. Western blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 and increases levels of γH2AX. Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening by amplification and subsequent sequencing of all coding exons of p53. All cell lines except for K562 and primary leukemia cells lacked mutations in the p53 gene, demonstrating that in Ph+ ALL PF-0477736 is highly effective also on p53 wild-type tumor cells. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on 3 treated Ph+ cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p < 0.05) of 211 genes including those involved in apoptosis and cell cycle (CEBPB, CUL1, Histone H1-H2A, 2B family clusters, Histone H4, DHX15, SNCB, FOS) and DNA damage, such as DNA-damage-inducible transcript 3 (DDIT3) and growth-arrest and DNA damage-inducible proteins GADD34 and GADD45a, suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in Ph+ ALL cells. In conclusion, for the first time we demonstrate in a large cohort of Ph+ ALL patients an over-expression of Chk1, providing a strong rational for its inhibition. In vitro treatment of Ph+ ALL cells with PF-0477736 alone resulted in reduction of inhibitory phosphorylation of Cdc25c, inhibition of proliferation and induction of biomarkers of DNA damage and apoptosis, suggesting that single-agent Chk1/2 inhibition may be an effective treatment strategy for Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Although impressive developments have been made in the treatment of Acute Lymphoblastic Leukemia (ALL) patients, the overall survival is still very poor. With the exception of novel therapeutic strategies based on monoclonal antibodies (Bi-specific T-cell engagers, BiTEs) or immunogenic cells (CART cells), the therapeutic approaches for adult ALL patients are still base on non-selective chemotherapy or on tyrosine kinase inhibitors (TKIs) for the patients harboring the BCR-ABL1 fusion transcript. In addition a large percentage of initial successfully treated patients frequently develop relapses. Thus there is a need to improve the efficacy of conventional therapies, in particular those related to TKIs and to DNA damaging agents, in order to reduce the off-target toxicity and avoid relapses. In the present study we evaluated the in vitro, ex vivo and in vivo efficacy of MK-1775, a specific Wee1 inhibitor, in single agent and in combination with different therapeutic agents normally used for the treatment of B-/T-ALL. We firstly started by evaluated the efficacy of the compound in single agent on a panel of human B and T ALL cell lines (n=8) and on primary cells isolated from the bone marrow of adult B-ALL patients (n=8). The inhibition of Wee1 deeply reduced the cell viability and the proliferation rate, induced the apoptosis and increased the DNA damages of both leukemic cell lines and primary cells. Further cell-cycle analysis showed that in leukemic cell lines the treatment increased the number of cell in late S and G2/M phase. Light microscopy analyses, looking for nuclei morphology, confirmed that MK-1775 increased the number of mitotic cells but it interfered with normal mitotic division (induction of aberrant mitosis as showed by the increment of DNA bridges and micro-nuclei). The effects of the compound on the cell cycle profile and on the G2/M checkpoint were confirmed also in immunoblotting analyses, by the increment of phospho-HH3(ser10) and of Myt1 (mitotic isoform), and by gene expression analysis looking to specific genes involved in the G2/M checkpoints (PrimePcr DNA damage assay, Biorad). In particular genes like GADD45A and CCNB1/CCNB2 were significantly up-regulated between treated and untreated samples. Finally using a T-ALL mouse model we evaluated the effect of MK-1775 in single agent. Although no significative differences were seen between treated and un-treated samples, due to a very aggressive phenotype of the disease (all animal died after only 18 days from the engraftment), molecular analyses confirmed that the treatment induced DNA damages (increase of H2A.X and p-Chk1 ser317) and inhibited Wee1 functionality (reduction of pCDC2) on leukemic blasts isolated from both spleens and bone marrows. To evaluate if the inhibition of the G2/M checkpoint could sensitize leukemic cells to the toxicity of antineoplastic drugs, Philadelphia-negative ALL cell lines and primary leukemic cells (n=9) where treated with increasing concentration of MK-1775 and increasing concentration of the nucleotide analogue, clofarabine. Statistical analyses (Combination index value) confirmed the synergy of the combination in the reduction of the cell viability, in the inhibition of the proliferation and in the induction of the apoptosis. Similar results were seen on Philadelphia-positive ALL cell lines and primary cells (n=3) combining the MK-1775 with the TKI, bosutinib. The simultaneously inhibition of the Wee1 and the BCR-ABL downstream pathway resulted in a synergic inhibition of the cell viability, reduction of the proliferation and induction of apoptosis. In our opinion the pre-clinical results of this study are the basis for a future clinical evaluation of MK-1775 for the treatment of ALL patients. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Speakers Bureau; BMS: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau.

The precise regulation of the iron-regulatory hormone hepcidin is essential to maintain body iron homeostasis: Hepcidin deficiency induces iron overload, and hepcidin excess results in anaemia. Mutations in the gene HFE2 cause severe iron overload and are associated with low hepcidin expression. Recent data suggest that HFE2 is a bone morphogenetic protein (BMP) co-receptor, and that the decreased hepcidin mRNA expression because of HFE2 dysfunction is a result of impaired BMP signalling ability. In this study, we identify a critical BMP-responsive element (BMP-RE) at position -84/-79 of the hepcidin promoter. We show that this element mediates HFE2-dependent basal hepcidin mRNA expression under control conditions. Unexpectedly, the mutation of the same BMP-RE element also severely impairs hepcidin activation in response to IL-6. These data uncover a missing link in the HFE2-mediated control of hepcidin expression and suggest that the BMP-RE controls hepcidin promoter activity mediated by HFE2 and inflammatory stimuli.

We showed that extended self-renewal of leukemia stem-cells (LSCs) is the consequence of the constitutive activation of the cell-cycle inhibitor p21. The pool of LSCs is markedly reduced in p21-/- APLs, and the residual LSCs hyperproliferate and accumulate massive DNA-damage (DD). Notably, p21 is indispensable to maintain the pool of LSCs in PML-RAR leukaemias (PML-RAR – p21-/- leukaemias are not transplantable in syngenic mice). It is not known whether p21 extends the self-renewal of LSCs by activating DD repair, restricting the cell-cycle or regulating their survival. Preliminary results from our lab showed that p21-/- APLs re-acquire the ability to initiate leukemogenesis when transplanted into immunodeficient mice (RAG) or syngeneic mice after γ-irradiation. In these conditions, the growing leukemias are indistinguishable from primary WT APLs and can be re-transplanted into immunodeficient, but not syngeneic mice, suggesting that expansion of p21-/- LSCs is controlled by the host immune-system. Further experiments suggested that p21-/- LSCs are cleared by T-cells since p21-/- APLs: i) initiate leukemias when transplanted into RAG1-/- mice (which lack B, T and NKT cells), but not in JHT mice (lacking only B-cells), and ii) do not grow in NOD/SCID mice when the recipients are injected with a T cell population primed with p21-/- blasts (e.g. obtained from the spleen of WT mice injected with p21-/- APLs), but not with non-primed T cells or T cells primed with WT blasts. T-cells primed with p21-/- blasts are also able to suppress the in vivo growth of wt APLs, but not of T-ALLs. We hypothesize that the immune-surveillance contributes to the elimination of DNA-damaged SCs, and that DNA-damaged LSCs have developed evasion strategies based on p21 activation. Citation Format: Maria Vittoria Verga Falzacappa, Alessandra Insinga, Pier Giuseppe Pelicci. Regulation of self-renewal in cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA33.

This work shows how the introduction of an organometallic group enhances and modifies the specificity of biologically active peptides. Ferrocene was chosen as an organometallic group because it has been shown to alter the pharmacodynamic profile of bioactive compounds. A comparison with the isosteric cobaltocenium group allows one to explore the influence of charge and redox potential on the biological activity of the conjugates. Arginine and tryptophan containing peptides H-WRWRWR-NH(2) and H-RWRWRW-NH(2) and the metallocene peptide bioconjugates [M]-C(O)-RWRWR-NH(2) and [M]-C(O)-WRWRW-NH(2), where [M]=[Co(Cp)(C(5)H(4))](+), [Fe(Cp)(C(5)H(4))] were prepared by solid-phase peptide synthesis (SPPS). They were purified by HPLC, characterized by ESIMS and NMR spectroscopy, and tested for antibacterial properties against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus using the minimum inhibitory concentration (MIC) test. In most cases, no metal-specific activity could be observed. However, the conjugate [Fe(Cp)(C(5)H(4))-C(O)-WRWRW-NH(2)] 6 was found to be particularly effective against the Gram-positive S. aureus. The activity of this metallocene-pentapeptide conjugate (7.1 microM) was even better than the 20 amino acid naturally occurring pilosulin 2, which was used as a positive control. Unlike all other compounds tested, which were most active against the Gram-negative E. coli strain, the ferrocene conjugate 6 was the only compound in this series that was most active against Gram-positive bacteria. Given the health concerns resulting from multidrug resistant S. aureus strains, the incorporation of metallocenes may provide a novel line of attack.

Hepcidin is a key iron-regulatory hormone produced by the liver. Inappropriately low hepcidin levels cause iron overload, while increased hepcidin expression plays an important role in the anemia of inflammation (AI) by restricting intestinal iron absorption and macrophage iron release. Its expression is modulated in response to body iron stores, hypoxia, and inflammatory and infectious stimuli involving at least in part cytokines secreted by macrophages. In this study we established and characterized IL6-mediated hepcidin activation in the human liver cell line Huh7. We show that the proximal 165 bp of the hepcidin promoter is critical for hepcidin activation in response to exogenously administered IL6 or to conditioned medium from the monocyte/macrophage cell line THP-1. Importantly, we show that hepcidin activation by these stimuli requires a STAT3 binding motif located at position –64/–72 of the promoter. The same STAT binding site is also required for high basal-level hepcidin mRNA expression under control culture conditions, and siRNA-mediated RNA knockdown of STAT3 strongly reduces hepcidin mRNA expression. These results identify a missing link in the acute-phase activation of hepcidin and establish STAT3 as a key effector of baseline hepcidin expression and during inflammatory conditions.

Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

In acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify the genomically clustered miR-181a and miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. In addition, the expression of the miR-181a/b-cluster was strongly reduced in bone marrow samples of APL patient while ATRA-based therapy. Furthermore, we showed the transcriptional induction of miR-181a and miR-181b by the APL-associated PML-RARα oncogene in vitro and in vivo. In PR9 cells, carrying a zinc-driven PML/RARα construct, and in PML/RARα-knock in mice the expression of the fusion gene leads to upregulation of the microRNA-cluster expression. Analysis of bone marrow samples of APL patients showed an enhanced expression of miR-181a and miR-181b in comparison to AML patient samples with normal karyotype, whereas other AML subgroups show no significant regulation. Based on siRNA experiments we could propose AP-1 and GATA-2 as potential co-activators for the PML/RARα-dependent regulation of the miR-181a/b-cluster. In functional studies in NB4 cells we observed after lentiviral knock down of miR-181a and miR-181b a significant reduction of colony size and number as well as proliferation rate. In contrast, transient overexpression of miR-181a and miR-181b led to an inhibition of ATRA-induced expression of the differentiation marker CD11b. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181a and miR-181b. In functional studies we showed that enforced expression of miR-181a and miR-181b reduces the protein level of RASSF1A by binding to the 3´UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. Overexpression of RASSF1A in NB4 cells strongly induced apoptosis. Additional, we could show by western blot that the miR-181a/b-cluster and RASSF1A modulate cell cycle via regulation of cyclin D1. In conclusion, we identified the miR-181a/b-cluster as an important player in the PML/RARα associated APL. Moreover, we firstly described the miR-181a/b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated microRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

The evolution of orthologous genes coding for β-defensin 2 (BD2) in primates has been subject to positive selection during the divergence of the platyrrhines from the catarrhines and of the Cercopithecidae from the Hylobatidae , great apes, and humans. Three peptides have been selected for a functional analysis of the effects of sequence variations on the direct antimicrobial activity: human BD2 (hBD2), Macaca fascicularis BD2 (mfaBD2), and a variant of the human peptide lacking Asp 4 , (−D)hBD2, which is characteristic only of the human/great ape peptides. hBD2 and mfaBD2 showed a significant difference in specificity, the former being more active towards Escherichia coli and the later towards Staphylococcus aureus and Candida albicans. Asp 4 in the human peptide appears to be important, as (−D)hBD2 was less structured and had a markedly lower antimicrobial activity. The evolution of β-defensin 2 in primates may thus have been driven, at least in part, by different environmental pressures so as to modulate antimicrobial activity.

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289 ]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.

Although improvements in knowledge of genetic basis of ALL have been made and many new drugs are now available, the prognosis for adult patients with ALL is still a challenge. Current therapies based on chemotherapy regimens or on tailored targets do not completely eradicate the leukemia clone and relapse is a predictable event. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), an ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Chk1 and Chk2 are serine/threonine kinases which following activation by a broad spectrum of damaged DNA coordinate the DNA damage response. By quantitative PCR, higher transcript levels of Chk1 were found in 8 leukemia cell lines and 54 newly diagnosed ALL cases compared to normal bone marrow mononuclear cells (p value < 0.001). Immunohistochemistry of formalin-fixed paraffin-embedded tissue samples collected from 60 ALL patients showed a diffuse positivity for Chk1, Chk2 and the phosphorylated forms of Chk1 (Ser345) in 51/54 (96%), 55/57 (94%) and 45/56 (80%) of the cases respectively, whereas 15/55 (27%) of ALLs stained for phosphorylated form of Chk2 (Thr68). Interestingly, in our ALL series, genomic damage was suggested by the nuclear labeling for γ-H2A.X molecule in 40/59 (68%) of samples. In B-/T-ALL cell lines (BV-173, SUP-B15, REH, NALM-6, NALM-19, MOLT-4, RPMI8402 and CCRF-CEM) inhibition of Chk1 resulted in a dose and time-dependent cytotoxicity (IC50 from 57.4 nM to 1423.0 at 24 hours), induction of apoptosis and increased DNA damage as demonstrated by increased levels of phosphorylated Chk1 (Ser317 and Ser345) and H2A.X (Ser139). Moreover, PF-00477736 efficiently triggered the Chk1-Cdc25-Cdc2 pathway with a decreased phosphorylation of Cdc25c (Ser216) and Cdc2 (Tyr15). Gene expression profiling analysis shed light on molecular mechanisms driven by PF-00477736 revealing a deregulation of genes involved in DNA damage checkpoint, cell cycle control and apoptosis. Among these, Gadd45a and Plk3 were confirmed by qPCR analysis up-regulated, whereas Cdk4 and Chk2 resulted down-regulated between treated and untreated samples. The efficacy of PF-00477736 as a single agent was confirmed ex-vivo in primary leukemic blasts separated from 14 ALL patients but not in in primary cultures of normal bone marrow mononuclear cells. Furthermore, in mice transplanted with T-lymphoid leukemia PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). In conclusion, in vitro, ex-vivo and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia and they provide a strong rationale for its future clinical investigation. ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, NGS-PTL project, Programma di ricerca Regione- Università 2010 -2012, Regione Emilia-Romagna, Bando “Ricerca Innovativa” (Prof. L. Bolondi). Citation Format: Ilaria Iacobucci, Andrea Ghelli Luserna Di Rorà, Maria Vittoria Verga Falzacappa, Claudio Agostinelli, Enrico Derenzini, Anna Ferrari, Cristina Papayannidis, Annalisa Lonetti, Simona Righi, Enrica Imbrogno, Claudia Venturi, Viviana Guadagnuolo, Federica Cattina, Emanuela Ottaviani, Maria Chiara Abbenante, Domenico Russo, Pier Luigi Zinzani, Stefano Pileri, Pier Giuseppe Pelicci, Giovanni Martinelli. In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia. [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 LB-105. doi:10.1158/1538-7445.AM2014-LB-105

Introduction: Chk1 and Chk2 are serine/threonine kinases that are involved in cell cycle control and DNA repair pathways. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1-negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5-2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of baseline genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. Moreover, in all sensitive cell lines Chk1/Chk2 inhibition increased number of γH2AX foci (western blot and immunofluorescence analysis) and phosphorylation of Chk1 (ser317 and ser345) representative of induced DNA damage. Functionally, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15). The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients with 36% of them having IC50 within 100-500 nM. By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells. Noteworthy, in mice transplanted with T-lymphoid leukemia, PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts after 24 hrs to elucidate the mechanisms of action of PF-0477736. Results showed a differential expression (p < 0.05) of genes involved in chromatin modification (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in leukemia cells. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007-2009. PF-0477736 provided by Pfizer. Citation Format: Ilaria Iacobucci, Andrea Ghelli Luserna Di Rorà, Maria Vittoria Verga Falzacappa, Enrico Derenzini, Anna Ferrari, Enrica Imbrogno, Cristina Papayannidis, Claudia Venturi, Annalisa Lonetti, Viviana Guadagnuolo, Federica Cattina, Emanuela Ottaviani, Mariachiara Abbenante, Domenico Russo, Pier Giuseppe Pelicci, Giovanni Martinelli. Inhibition of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) is a novel therapeutic strategy in B- and T-Acute Lymphoblastic Leukemia (ALL). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2013-705

Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, we investigated the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in B-ALL and we determined potential biomarkers of functional inhibition. BCR-ABL1-positive (BV-173, SUPB-15) and negative cell lines (NALM6, NALM19, REH) were incubated with increasing concentrations of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1-1.5 µM, with BV-173 being the most sensitive, while NALM6 the most resistant (WST-1 assay, Roche). Consistent with the viability results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in all cell lines. Western Blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry, and increases phosphorylation of γH2AX Ser139, a marker of DNA damage. The efficacy of PF-0477736 was thereafter confirmed in primary blast cells from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50 ranged from 0.1-0.5 µM at 24 hours); good responders, 36% (IC50 ranged from 0.6-1 µM at 24 hours); poor responders, 18% (IC50 > 1 µM at 24 hours). Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening of all coding exons of p53. All cell lines and primary leukemia blasts lacked mutations, demonstrating that in B-ALL the sensitivity to PF-0477736 is independent of p53 status. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p < 0.05) of genes involved in apoptosis and cell cycle (e.g. CEBPB, CUL1, Histone H1-H2A, 2B family clusters) and DNA damage (DDIT3, GADD34 and GADD45a), suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in B-ALL cells. In conclusion, for the first time we demonstrated the efficacy of PF-0477736 in vitro models of B- ALL, suggesting that single-agent Chk1/2 inhibition may be further evaluated in clinical trials. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007-2009. PF-0477736 provided by Pfizer. 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 1772. doi:1538-7445.AM2012-1772

Abstract 1361 In the acute promyelocytic leukemia (APL) bearing the translocation t(15;17), all-trans-retinoic acid (ATRA) treatment induces granulocytic maturation and complete remission of leukemia. Several factors are involved in the formation of the leukemic phenotype. Latest studies identified microRNAs as critical players in this network. In a micro array based microRNA screen we could identify miR-181b as downregulated in the APL cell line NB4 by treatment with pharmacological doses of ATRA. Additionally, we showed the transcriptional induction of miR-181b by the APL-associated PML-RARα oncogene which is released after treatment with ATRA. The overexpression of miR-181b by microRNA mimics leads to an inhibition of ATRA induced granulocytic differentiation. Furthermore, the downregulation of miR-181b by locked nucleic acids (LNAs) causes the induction of granulocytic transcription factor C/EBPβ. In a PML/RARα knock in mouse model we could show the enhanced expression of miR-181b. Also, APL patient samples show a significant enhanced expression of miR-181b in comparison to AML patient samples with normal karyotype. In addition, the miR-181b expression was strongly reduced in APL patient samples after ATRA treatment. In a microRNA target search we identified the novel ATRA regulated tumor suppressor RASSF1A as a putative target of miR-181b. In functional studies we showed that enforced expression of miR-181b reduces the protein level of RASSF1A by binding to the 3'UTR of RASSF1A mRNA. Accordingly, RASSF1A protein was enriched after knock down of miR-181b. The role of RASSF1A in ATRA induced differentiation was verified by knock down of RASSF1A protein by specific siRNA: Here we could show the reduction of ATRA induced CD11b expression. In conclusion, we identified miR-181b as a new player in the PML/RARα associated APL. Moreover, we firstly described the miR-181b target RASSF1A as a crucial factor in the ATRA activated granulocytic differentiation program in APL. Our data reveal the importance of deregulated miRNA biogenesis in cancer and may provide novel biomarkers and therapeutic targets in myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.