Viviana Guadagnuolo

IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia.

Background Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. Principal Findings Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. Conclusions Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.

The PAX5 gene is frequently rearranged in BCR-ABL1-positive acute lymphoblastic leukemia but is not associated with outcome. A report on behalf of the GIMEMA Acute Leukemia Working Party

Background Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric patients with acute lymphoblastic leukemia. So far the occurrence of PAX5 alterations and their clinical correlation have not been investigated in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Design and Methods The aim of this study was to characterize the rearrangements on 9p involving PAX5 and their clinical significance in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Eighty-nine adults with de novo BCR-ABL1-positive acute lymphoblastic leukemia were enrolled into institutional (n=15) or GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto) (n=74) clinical trials and, after obtaining informed consent, their genome was analyzed by single nucleotide polymorphism arrays (Affymetrix 250K NspI and SNP 6.0), genomic polymerase chain reaction analysis and re-sequencing. Results PAX5 genomic deletions were identified in 29 patients (33%) with the extent of deletions ranging from a complete loss of chromosome 9 to the loss of a subset of exons. In contrast to BCR-ABL1-negative acute lymphoblastic leukemia, no point mutations were found, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1-positive acute lymphoblastic leukemia. The deletions were predicted to result in PAX5 haploinsufficiency or expression of PAX5 isoforms with impaired DNA-binding. Deletions of PAX5 were not significantly correlated with overall survival, disease-free survival or cumulative incidence of relapse, suggesting that PAX5 deletions are not associated with outcome. Conclusions PAX5 deletions are frequent in adult BCR-ABL1-positive acute lymphoblastic leukemia and are not associated with a poor outcome.

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

IL-17/IL-10 double-producing T cells: new link between infections, immunosuppression and acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is an incurable disease with fatal infections or relapse being the main causes of death in most cases. In particular, the severe infections occurring in these patients before or during any treatment suggest an intrinsic alteration of the immune system. In this respect, IL-17-producing T helper (Th17) besides playing a key role in regulating inflammatory response, tumor growth and autoimmune diseases, have been shown to protect against bacterial and fungal pathogens. However, the role of Th17 cells in AML has not yet been clarified.MethodsT cell frequencies were assessed by flow cytometry in the peripheral blood of 30 newly diagnosed AML patients and 30 age-matched healthy volunteers. Cytokine production was determined before and after culture of T cells with either Candida Albicans or AML blasts. Statistical analyses were carried out using the paired and unpaired two-tailed Student’s t tests and confirmed with the non parametric Wilcoxon signed-rank test.ResultsA strong increase of Th17 cells producing immunosuppressive IL-10 was observed in AML patients compared with healthy donors. In addition, stimulation of AML-derived T cells with a Candida albicans antigen induced significantly lower IFN-γ production than that observed in healthy donors; intriguingly, depletion of patient Th17 cells restored IFN-γ production after stimulation. To address the role of AML blasts in inducing Th17 alterations, CD4+ cells from healthy donors were co-cultured with CD33+ blasts: data obtained showed that AML blasts induce in healthy donors levels of IL-10-producing Th17 cells similar to those observed in patients.ConclusionsIn AML patients altered Th17 cells actively cause an immunosuppressive state that may promote infections and probably tumor escape. Th17 cells could thus represent a new target to improve AML immunotherapy.

The human Smoothened inhibitor PF-04449913 induces exit from quiescence and loss of multipotent Drosophila hematopoietic progenitor cells.

The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.

Chromothripsis in acute myeloid leukemia: biological features and impact on survival

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study define incidence of chromothripsis in 395 newly-diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p=.002), ELN high risk (HR) (p<.001), lower white blood cell (WBC) count (p=.040), TP53 loss and/or mutations (p<.001) while FLT3 (p=.025) and NPM1 (p=.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p<.001) compared with HR patients (p=.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e. TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, 17. CBA. FISH showed that chromothripsis is associated with marker, derivative and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression—and consequently, H3K36 trimethylation—was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

A Case Report of Acute Myeloid Leukemia and Neurofibromatosis 1

We report a case of a 65-year old patient affected by neurofibromatosis 1, documented by the presence of germ-line mutation on the NF1 gene, who developed various hyperproliferative malignant and benign diseases. He was brought to our attention for the diagnosis of acute myeloid leukemia revealed by major fatigue and dyspnea. The disease characteristics at diagnosis were hyperleukocytosis and complex karyotype with the inversion of the chromosome 16, classifying as a high-risk leukemia. The association between leukemia and neurofibromatosis 1 is controversial and needs to be further investigated. Nevertheless, such patients present a wide number of comorbidities that make therapeutic strategies most difficult.

Abstract 906: Gas1 and Kif27 genes are strongly up-regulated biomarkers of Hedgehog inhibition (PF-04449913) on leukemia stem cells in Phase I Acute Myeloid Leukemia and Chronic Myeloid Leukemia treated patients

Hedgehog (Hh) pathway activation contributes to leukemia development and growth, and that targeted pathway inhibition is likely to offer an efficient therapeutic opportunity. PF-04449913, a Hh pathway inhibitor, is a new selective and potent inhibitor of leukemia self-renewal and is currently being evaluated in phase I clinical trials. In order to identify new potential clinical biomarkers for the PF-04449913, we studied CD34+ leukemia stem cell population (LSC) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies. This experimental clinical trial enrolled Myelofibrosis (MF), MDS, blastic phases CML, chronic myelomonocytic leukemia (CMML) and AML patients (pts). We were able to collect and separate highly purified (98%) bone marrow CD34+ cells from 5 AML, 1 MF and 2 CML pts by immunomagnetic separation, and analysed them for gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Clustering of their expression profiles showed that mostly genes differentially expressed are mainly related to Hh signaling, this providing further evidences that PF-04449913 really therapeutically targets the Hh pathway. Regarding genes involved in Hh signaling pathway, Gas1 and Kif27 were strongly upregulated (fold change 1.0947 and 1.12757 respectively; p-value 0.01 and 0.02 respectively) in CD34+ LSC after 28 days exposure to PF-04449913 as compared to baseline, suggesting these two genes have potential as new biomarkers of activity. GAS-1 is a Sonic Hedgehog (Shh)-binding protein; it acts to sequester Shh and inhibit the Shh signalling pathway. Kif27 mainly acts as a negative regulator in the Hh signaling pathway, and inhibits the transcriptional activator activity of Gli1 by inhibiting its nuclear translocation. Other genes were differentially expressed after ‘ex- vivo’ treatment with PF-04449913 as compared to baseline: we observed a down regulation of Bcl2 (fold change -1.03004), ABCA2 (fold change -1.08966), LEF1 (fold change -1.28457), Gli1 (fold change -1.0775), Smo (fold change -1.07702), and an upregulation of Gli2 (fold change 1.08191). Conclusions: This data demonstrates that PF-04449913 specifically targets the Hh Pathway in CD34+ cells, suggesting that Hh inhibition may impair leukemia stem cell maintenance. In addition, we identify several new potential biomarkers (e.g. Gas1 and KIF27). Taken together, these data may be useful for pts selection strategies and subsequent eradication of the LSC. Acknowledgments: Work supported by Pfizer, European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. 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 906. doi:1538-7445.AM2012-906

Abstract 2723: The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia

Different Checkpoint kinase inhibitors (Chk-i) have been developed to increase the cytotoxic effect of genotoxic agents inhibiting the key elements of the DNA damage response (DDR) pathways. Our group has already showed the efficacy of this class of compounds in single agent in different in vitro/ex vivo/in vivo studies for the treatment of acute lymphoblastic leukemia (ALL). The aim of the study was to evaluate the efficacy of a Chk1/Chk2 inhibitor in combination with the topoisomerase II inhibitor doxorubicin for the treatment of ALL. Firstly we evaluate the efficacy of doxorubicin on human B (NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathways. Cells were treated with doxorubicin (0.25-2.5 uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. The induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was also evaluated in all the cell lines. Due to the inhibitory effect of the compound on the topoisomerase II enzyme and due to the activation of the cell cycle checkpoint, cells were arrested in G2/M phase. Then the effectiveness of the Chk-i as a chemo-sensitizer agent was evaluated. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk-i (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a synergistic effect in term of reduction of the cell viability and induction of apoptosis. The effect of the combination was also analyzed using western blot looking for specific marker of activation of the DDR pathway showing the same synergistic effect. Moreover the effect of the combination on cell cycle profile was evaluated using a double staining Pi/Anti-phospho-Histone H3 ser10 (marker of mitosis). Cell lines were pre-treated for 18 hours with doxorubicin and then with the Chk-i for different time points (1, 2, 3, 6 and 9 hours). The treatment with Chk-i removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells positive for the mitotic marker p-HH3 (ser10) and increasing the percentage of cells in sub-G1 phase. In our opinion the combination between the Chk1 inhibitor,LY2606368, and the topoisomerase II inhibitor, doxorubicin, could be a promising strategy for the treatment of B/T-ALL. Supported by ELN, AIL, AIRC, progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinelli. The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2723.