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Martinelli: Roche: Consultancy; ARIAD: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy.

Acute lymphoblastic leukemia (ALL) cells respond to chemotherapy, or more generally to DNA damages, by activating different DNA Damage Response (DDR) pathways. DDR-pathways regulate cell cycle progression and DNA damages repair. Molecular and functional alterations in key DDR-related genes drastically affect the effectiveness of DNA-damaging treatments in cancer cells. For this reasons selective DDR-inhibitors have been developed in order to sensitize cancer cells against conventional chemotherapy. Despite the proven efficacy of DDR-inhibitors in cancer treatment, only few studies have highlighted the biological consequences the prolonged inhibition of DDR-pathways in cancer cells. We hypothesized that the protracted inhibition of the DDR pathways may generate resistant clones characterized by an increased genetic instability. The aim of the study was to evaluate biological consequences of the prolonged inhibition of two crucial DDR-related kinase such as cell cycle checkpoint kinase 1 (Chk1) and 2 (Chk2) in B cells ALL. In particular, we investigated the consequences of Chk1/Chk2 inhibition in term of increase of genetic instability and in term of responsiveness to chemotherapy agents. Starting from B-ALL cell line NALM-6, we generated a resistant model (hereafter referred as N6R-PF8) by treating the parental cells with increasing concentration of PF-00477736 (Chk1/Chk2 inhibitor) for more than a year and increasing the IC50 value of 10-folds. From a molecular point of view, the N6R-PF8 accumulated significant molecular alterations. SNP microarray analysis highlighted different alterations in DDR-related genes and, in particular, in the ATM/CHK2 pathway. Three regions in copy number LOSS (CN=1) containing several genes involved in cell cycle checkpoint regulation (ATM and NPAT) and in the apoptosis (BIRC2, CASP1 and CASP5) were detectable only in NALM-6 parental cell lines and were copy number neutral in the resistant model. Immunoblotting analysis confirmed that in N6R-PF8 cells the ATM/CHK2 and ATR/CHK1 down-stream pathways were significant over-expressed and activated in comparison to the parental cell. Whole exome sequencing analysis showed that the two cell lines were characterized by different mutational profiles and that N6R-PF8 cells harboured significantly more genetic alterations in comparison with NALM-6 cells. Interestingly, crucial genes involved in DNA repair pathway (MLH3, NBN, POLD1and PMS2) have been found altered only in N6R-PF8 cells. From a functional point of view, the molecular alterations characterizing the N6R-PF8 significantly compromised the cytotoxicity of PF-00477736 and of different DNA damaging agents in comparison to parental cells. Furthermore, the treatment with ATR/ATM inhibitor restored the sensitivity of N6R-PF8 to PF-00477736 and to different chemotherapy agents. In this scenario the level of expression of these two kinases seems to correlate with the sensitivity to DNA damaging agents and to PF-00477736. Finally, we confirmed that the protracted inhibition of crucial DDR-related kinase may increase the overall genetic instability in ALL cells and compromise the efficacy of DNA damaging based therapies.

Cavo: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Novartis: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad/Incyte: Consultancy; Amgen: Consultancy.

Background: The heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL patients (pts) that doesn't have the most recurrent adult rearrangements (BCR-ABL1t(9;22); TCF3-PBX1t(1;19); MLL-AF4t(4;11)) are collectively referred to as “triple negative” (Ph-/-/-) ALL. CRLF2is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Clustering and biological characterization of Ph-/-/- ALL (that represents 61% of adult B-ALL; Roberts KG, J Clin Oncol. 2016), considering CRLF2overexpression event, in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) were performed on 55 Ph-/-/- ALL, 29 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized with the Expression Console Software. Successively we cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in the top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (TruSight Pancancer-Illumina; MLPA and/or dMLPA-MRC-Holland; SNP Array-Affymetrix; 454 Junior-Roche and PCR). Results: Clustering our Ph-/-/- gene expression data using the impact of the 10 single genes in our cohort, we could identify a defined 2-clusters-subdivision (Gr1 and Gr2; Fig 1A). The Gr2 is characterized by CTGF, CRLF2and CD200(Gr2=3C-up; Fig 1B) overexpression and it represents 14.1% of all B-ALL. The Gr2 GEP is similar to Ph+ one. Fusion copy number alteration and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7Rmutations or deletion), that mainly affect JAK-STAT pathway. Also IKZF1and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. Molecular characterization shed light on a very heterogeneous scenario especially in the group 1, suggesting the need of a more discerning clustering for this group. In spite of the small number of cases is required, preliminary Gr1 subclustering discerns MLLr and ZNF384 gene expression subgroups. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upregulated in the group1 and CDK6 in the Gr2. CRLF2and CD200immunoblotting and CD200immunohistochemistry preliminary analyses suggest that protein expression of CRFL2 and CD200 are higher in Gr2 in comparison to Gr1. Conclusions: we identified a new signature, related to CRLF2high expression, to classify Ph-/-/- ALL B-based on 10 genes. 3C-up represents 14.1% of all B-ALL and it is characterized by a) high co-expression of three main genes: CRLF2, CTGFand CD200;b) IKZF1deletion; c) JAK-STAT pathway mutations/fusions/deletions.Gr1 represents 46.9% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identify new potential therapeutic targets with available drug (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; tyrosine kinase inhibitors already effective on Ph+ and Ph-like) to test. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.

Introduction: “B-others” is an heterogeneous Ph negative adult ALL subtype that continue to have a poor prognosis and need to be better molecular defined by identifying genetic alterations that predict the risk of treatment failure and developing novel and targeted therapies.

The anthracycline antibiotic, doxorubicin, inhibits the topoisomerase II enzyme resulting in the abrogation of DNA synthesis and in the induction of DNA damages. This compound is normally used in several chemotherapy schedules for the treatment of leukemia. From a biological point of view, it has been reported that doxorubicin-treated cells arrest in G2/M phase as a consequence of the induction of DNA damages and of the induction of the DNA damage response (DDR) pathway. The activation of the G2/M checkpoint is regulated by three kinases: the cell-cycle checkpoint kinase 1 (CHK1) and 2 (CHK2) and WEE1. The activity of these kinases, which act as important tumor-suppressors on normal cells, can protect tumor cells from genotoxic agents. During the last years several studies have showed the efficacy of selective DDR inhibitors in single agent or in combination with DNA damaging agents. On these bases, the aim of the study was to evaluate the effects of a selective CHK1/CHK2 inhibitor (hereafter CHKi) in combination with doxorubicin (hereafter Doxo) on a panel of B and T acute lymphoblastic leukemia (ALL) cell lines (NALM-6, NALM19, RPMI-8402, MOLT-4 and CCRF-CEM). To this purpose we firstly evaluate the effect of Doxo in single agent on B-/T-ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathway. Cells were treated for 24/48 hours with increasing concentration of Doxo (0.25, 0.5 and 1 uM) showing a dose-dependent reduction of the cell viability, induction of apoptosis and increment of cells arrested in S or G2/M phases of the cell cycle. No differences in term of sensitivity to Doxo were seen between ALL cell lines with wildtype or mutated TP53. Then we evaluated the chemosensitizer efficacy of the CHKi against Doxo on the panel cell lines. To this purpose cells were treated simultaneously with increasing concentration of Doxo in combination with the CHKi for 24/48 hours and then the combination index of the two compounds was evaluated using Compusy Software. Only an additive effect in term of reduction of the cell viability was seen when the two compounds were added simultaneously. Based on these results we performed different schedules of combinations firstly treating with Doxo and then with CHKi or vice versa,defining the most effective schedule when cells were treated firstly with the CHKi (1 hour) and then with Doxo (24 hours). In this experimental setting the strongest effect was seen on MOLT-4 (TP53 mutated) cell line in which the pre-treatment with the CHKi impaired the G2/M checkpoint induced, later, by Doxo. Immunoblotting analyses showed that, in MOLT-4, the effect on the cell viability was associated with an increment of DNA damage (gH2AX) which was significantly higher in the combination. Interestingly on MOLT-4 the treatment with Doxo in single agent activated the cell cycle inhibitor p21 but not in the sample treated with the combination. On NALM-6 cell line (TP53 wildtype), the treatment with Doxo alone or in combination with CHKi activated p21. This data was associated with the poor effectiveness of the combination on NALM-6 cell line (poor reduction of cell viability and of induction of gH2AX). Although further experiments should be performed, first of all to investigate the role of p21 on the G2/M checkpoint, in our opinion the combination between Doxo and CHKi could be a promising strategy for the treatment of ALL. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, HARMONY project.

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.

Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

Background: Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting.

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 vivoand in vivoefficacy 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.

Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:BMS: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Martinelli:ROCHE: Consultancy; Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

Nowadays the effectiveness of the treatments for adult Acute Lymphoblastic Leukemia (ALL) patients is still inadequate and frequently many patients after years of response to treatments develop relapses. Thus there is a need to find novel targets for specific therapies and to maximize the effect of the actual treatments. Recently different Checkpoint Kinase (Chk)1/Chk2 inhibitors has been assessed for the treatment of different type of cancers but only few studies have been performed on hematological diseases. We evaluated the effectiveness of the Chk1 inhibitor, LY2606368, as single agent and in combination with tyrosine kinase inhibitors (imatinib and dasatinib) or with the purine nucleoside antimetabolite clofarabine in B-/T- acute lymphoblastic leukemia (ALL) cell lines and in primary blasts. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were incubated with increasing concentrations of drug (1-100 nM) for 24 and 48 hours and the reduction of the cell viability was evaluated using WST-1 reagent. LY2606368 deeply reduced the cell viability in a dose and time dependent manner in all the cell lines, with the BV-173 (6.33 nM IC50 24hrs) and RPMI-8402 (8.07 nM IC50 24hrs) being the most sensitive while SUP-B15 (61.4 nM IC50 24hrs) and REH (96.7 nM IC50 24hrs) being the less sensitive cell lines. Moreover the sensitivity to the compound was no correlated with the different sub-type of ALL or with the mutational status of p53, which is a marker of the functionality of the G1/S checkpoint. The cytotoxic activity was confirmed by the significant increment of apoptosis cells (Annexin V/Propidium Iodide), by the increment of gH2AX foci and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). To understand the relationship between the activation of apoptosis and the effect on cell cycle and to identify hypothetical mechanisms of death, different cell cycle analyses were performed (Propidium Iodide staining). The inhibition of Chk1, deeply changed the cell cycle profile. Indeed in all the cell lines the percentage of cells in S phase and in G2/M phase were reduced by the treatment while the numbers of cells in sub-G1 and G1 phase were increased. The hypothetical function of LY2606368 as a chemosensitizer agent was evaluated combining the compound with different drugs normally used in clinical trials. For each drugs the combination strongly reduced the cell viability when compared to the cytotoxic effect of the single drugs. Moreover the combination showed an additive efficacy in term of induction of DNA damages as showed by the increase number of gH2AX foci and the activation of pChk1 (ser 317). The results found on the cell lines were confirmed also using primary leukemic blast isolated from adult Philadelphia-positive ALL patients. Indeed LY2606368 as single agent or in combination with the Tki, imatinib, was able to deeply reduce the cell viability and to induce DNA damages (gH2AX foci). In conclusion LY2606368 showed a strong cytotoxic activity on B-/T-All cell lines and primary blasts as single agent and in combination with other drugs. In our opinion this data are the basis for a future clinical evaluation of this compound in the treatment of leukemia.

Acute Myeloid Leukemia (AML) is a highly heterogeneous disease and a complex network of events contribute to its pathogenesis. Chromosomal rearrangements and fusion genes have a crucial diagnostic, prognostic and therapeutic role in AML. A recent RNA sequencing (RNAseq) study on 179 AML revealed that fusion events occur in 45% of patients. However, the leukemogenic potential of these fusions and their prognostic role are still unknown.

Genomic instability and constitutive activation of the DNA damage response (DDR) pathway has been recently described in models of aggressive myc-driven lymphoid malignancies. The MYC oncogene has been reported to induce genomic instability by a mechanism involving replication stress. On the other hand, MYC is overexpressed in a fraction of diffuse large B-cell lymphomas (DLBCLs), and its overexpression has been reported to be associated with poor prognosis. The checkpoint kinases 1 (CHK1) and 2 (CHK2), are serine-threonine kinase involved in the DDR pathway. DDR activation triggers the phosphorylation of the histone H2AX at ser 139, a known marker of DNA damage and genomic instability. The correlation between genomic instability, MYC expression, and prognosis has not been investigated yet in DLBCL.

Genomic instability and constitutive activation of the DNA damage response (DDR) pathway has been recently described in models of aggressive myc-driven lymphoid malignancies. The MYC oncogene has been reported to induce genomic instability by a mechanism involving replication stress. On the other hand, MYC is overexpressed in a fraction of diffuse large B-cell lymphomas (DLBCLs), and its overexpression has been reported to be associated with poor prognosis. The checkpoint kinases 1 (CHK1) and 2 (CHK2), are serine-threonine kinase involved in the DDR pathway. DDR activation triggers the phosphorylation of the histone H2AX at ser 139, a known marker of DNA damage and genomic instability. The correlation between genomic instability, MYC expression, and prognosis has not been investigated yet in DLBCL.Immunohistochemistry (IHC) for phospho (γ) H2AX, pCHK1, pCHK2 was performed in tissue microarrays (TMAs) from 97 consecutive patients treated at our Institution between 2004 and 2011 with R-CHOP/CHOP-like regimens, with available paraffin embedded tissue from initial diagnosis. Moreover, to evaluate the therapeutic potential of DDR pathway inhibition in DLBCL, the DLBCL cell lines HBL-1, U2932, TMD8, SUDHL-6, BJAB, SUDHL-4 and primary DLBCL cells were incubated with the CHK inhibitor PF-0477736 (Pfizer).In the TMA study 57% of patients (n=55) displayed high levels of basal γH2AX (>30% of positive cells), 55% (n=53) displayed pCHK1/pCHK2 activation and of note all DLBCL cell lines showed detectable baseline activation of CHK1/CHK2 and/or H2AX phosphorylation, by western immunoblotting. γH2AX positive cases distributed equally in germinal center (GC) and in non GC DLBCLs, and were significantly associated with MYC expression (p<0.01). Five-year survival rate was 70% vs 41% for γH2AX-low and γH2AX-high patients respectively (p=0.01). Factors significantly related to the outcome in multivariate analysis were International Prognostic Index (IPI) score and γH2AX expression. Remarkably the prognostic significance of γH2AX was particularly evident in the low risk IPI group (0-2 risk factors), identifying a subgroup characterized by worse outcome (54% 5-year OS). In the in vitro study a significant growth inhibition (WST-1 assay), was evident after 48 hrs in all cell lines (IC50 10-230 nM). PF-0477736 25-500 nM induced cell death by apoptosis (annexin V- propidium iodide staining) in a time and dose dependent manner. Notably PF-0477736 demonstrated activity also in primary DLBCL cells (IC 50 of 50-500 nM, 24 hrs). We observed inhibition of phosphorylation of the downstream target CDC25c (ser 216), coupled with a marked increase in γH2AX ser 139 and CHK1 phosphorylation (ser317 and 345) following treatment.A significant fraction of DLBCLs shows high levels of inherent genomic instability; the DDR activation marker γH2AX is a poor prognostic predictor in DLBCL and interestingly is significantly associated with MYC expression. DDR inhibition resulted to be highly effective in DLBCL cell lines and primary DLBCL cells; on treatment modifications of CHK1 and H2AX phosphorylation could be useful biomarkers of CHK inhibitors activity. These data provide strong rationale for targeting the DDR pathway and for clinical investigation of CHK inhibitors in DLBCL.No relevant conflicts of interest to declare.