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Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F, and MPLW515L, or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK–mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2—Wilms' tumor 1 (WT1)–binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically.Significance:TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase–positive malignant hematopoietic cells to PARP inhibitors.

Supplementary Methods and Results

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F, and MPLW515L, or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK–mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2—Wilms' tumor 1 (WT1)–binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Significance: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase–positive malignant hematopoietic cells to PARP inhibitors.

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. Graphical abstract • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

The DNA damage response (DDR) pathway is frequently deregulated in cancer and it represent an attractive therapeutic opportunity. In acute myeloid leukemia (AML), different mechanisms of DDR deregulation have been identified, but a systematic investigation on DDR alterations is missing. To understand how the DDR pathways contribute to leukemogenesis, we studied the gene expression and mutational profiles of 274 DDR genes by analysing 539 AML cases profiled by whole genome (WGS) and RNA sequencing. WGS data were used to identify mutations in genes of the DDR and in a panel of genes known to be mutated in AML (n=73). Transcriptomic data were analysed through unsupervised clustering, differential expression and enrichment analysis. We detected 150 single nucleotide variants (SNVs) in 130 patients (24%, average 0.3 SNVs/case). Genes mutated in more than 1% of cases were ATM, BLM, BRCA2, POLG and POLQ. The most frequently altered pathway was the homologous recombination/Fanconi Anemia (HR) pathway (29%), followed by the genes that coordinates the DDR pathway (20%). We detected a trend toward mutual exclusivity between mutations in TP53 and mutations in genes of HR pathway or the genes that coordinates the DDR pathway (adj-p Supported by the Torsten Haferlach-Leukämiediagnostik-Stiftung and AIRC IG 2019 (project 23810). Citation Format: Antonella Padella, Stephan Hutter, Wencke Walter, Constance Baer, Irene Azzali, Andrea Ghelli Luserna Di Rorà, Martina Ghetti, Lorenzo Ledda, Matteo Paganelli, Claudia Haferlach, Wolfgang Kern, Giorgia Simonetti, Giovanni Martinelli, Torsten Haferlach. Genomic and transcriptomic profiles of DNA damage response genes in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5788.

Non-coding (nc)RNAs, including circular (circ)RNAs, contribute to tumor development and progression. Several ncRNAs were shown to affect the onset, prognosis, and treatment of acute myeloid leukemia (AML) in the past years. The human Plasmacytoma Variant Translocation 1 (PVT1) gene maps on the long arm of chromosome 8 (8q24), in the same genomic region hosting MYC and encoding for 83 linear (PVT1, lncipedia.org) and 26 high-confidence circular isoforms (circPVT1, www.circbase.org). The most common isoform of circPVT1 is a product of back-splicing of 410 nt and contains the whole exon 2 of PVT1 in a closed loop-like structure (hsa_circ_0001821). The study aims to investigate the role of PVT1 isoforms and circPVT1 in AML. Firstly, we focused on the various PVT1 isoforms and their differential expression in leukemia. Fourteen out of the 83 linear isoforms are expressed in the hematopoietic tissues (lymph node and white blood cells, www.noncode.org), and 6 of them were detectable in AML cell lines, including the t(8;21) KASUMI-1 and the NPM1-mutated OCI-AML3 models, together with circPVT1. We designed two antisense-oligonucleotides (ASOs), mapping on common exonic region and targeting the linear isoforms expressed in OCI-AML3 and KASUMI-1 cells, and one ASO spanning the junction region of circPVT1. ASOs-mediated knockdown (KD) showed a relevant decrease of PVT1 signals, especially by ASO combination, and circPVT1 level using the specific ASO in both cell lines, under normoxia and hypoxia (1% O2). The downregulation led to a significant decrease in cell growth, but, interestingly, only circPVT1-KD induced apoptosis under both conditions in OCI-AML3. To further investigate the biological consequences of circPVT1-KD, we performed RNAseq assays. Data analysis was performed by pseudo alignment of paired-end reads to the human transcriptome, then counted with the Kallisto tool. Differential expression analysis of single isoforms was performed with the Sleuth tool on normalized transcript per million. We identified a core of 644 and 838 commonly regulated genes by circPVT1 in both cell lines under normoxia and hypoxia, respectively. Pathway analysis (performed by EnrichR) revealed that these genes are involved not only in the RNA regulatory pathways, as expected according to circRNA functions, but also in metabolic (e.g., KDM3A, GPI, NFKBA, RBM3, XBP1) and DNA damage response (e.g., PIDD1, MUC1, BCLAF1, BABAM2) pathways, opening a new scenario for synthetic lethality approaches. In conclusion, our findings show that silencing of circPVT1 or the predominantly expressed PVT1 isoforms dampens leukemia cell growth, indicating a role in AML pathogenesis, and suggest that targeting them may have therapeutic potentials in AML. Citation Format: Martina Ghetti, Antonella Padella, Eugenio Fonzi, Lorenzo Ledda, Andrea Ghelli Lusernadi Rorà, Matteo Paganelli, Doron Tolomeo, Clelia Tiziana Storlazzi, Giovanni Martinelli, Giorgia Simonetti. circPVT1 and linear PVT1 isoforms regulate cell growth, metabolic and DNA damage response related gene signatures in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1555.

Currently, the role of autophagy in acute lymphoblastic leukemia (ALL) is not fully understood however it has been linked to drug resistance. Several autophagy modulators have proven their efficacy in single agents or as chemo-sensitizer in ALL. Recently, the 3rd generation BCR-ABL1 inhibitor, ponatinib, has been showed to promote autophagy in chronic myeloid leukemia (CML) cells and the combination between ponatinib and hydroxychloroquine (HCQ) has proven its efficacy in neuroblastoma cells. Here, we tested the efficacy of combining different autophagy modulators (ponatinib, HCQ and rapamycin) against ALL cell lines and primary leukemic ALL cells. Firstly, we established the IC50 values of a panel of Philadelphia (Ph)-positive/negative B-/T-ALL cell lines (n=6) treated with ponatinib, HCQ and rapamycin in single agent. Then, using sub-toxic concentrations, we evaluated the efficacy of the combinations HCQ+Ponatinib and HCQ+Rapamycin in the reduction of the cell viability after 24, 48 and 72 hours of simultaneous treatment. Combination index analyses showed a synergic time and dosage-dependent reduction of the cell viability in all the cell lines treated with HCQ+Ponatinib. The combination HCQ+Rapamycin resulted synergic only in Ph-negative ALL cell lines. These results were confirmed by the significant induction of apoptosis (Annexin V and/or Caspase-3 staining) in the samples treated with HCQ+Ponatinib. The two combinations also significantly reduced the proliferation capacity of ALL cell lines in comparison with controls and single treatments. Moreover, the efficacy of the two combinations was confirmed on primary leukemic blasts isolated form adult ALL patients(n=6). Indeed, a significant induction of apoptosis was seen in samples treated with the two combinations in comparison with single treatments. To deeper understand the mechanism of action of the combinations we evaluated LC3A/B expression by flow cytometry. Interestingly, the expression of LC3A/B was modified heterogeneously among the different ALL cell lines and did not correlate with the response in term of cell viability or induction of apoptosis. Light microscopy analyses showed a significant increment in the number and diameter of autophagy vesicles in both ALL cell lines and primary leukemic ALL cells treated with HCQ+Ponatinib in comparison with single treatments. Interstingly, no significant effect on autophagy vesicles number or diameter was seen in the samples treated with HCQ+Rapamycin in comparison with single treatments. In conclusion, our findings show that the identified combinations effectively inhibit cell viability and induce apoptosis in Ph-positive/negative B-/T-ALL cells, suggesting the fundamental role of autophagy regulation in ALL survival. Citation Format: Andrea Ghelli Luserna Di Rora, Eugenio Fonzi, Lorenzo Ledda, Roberta Napolitano, Anna Ferrari, Antonella Padella, Martina Ghetti, Maria Teresa Bochicchio, Mouna Jandoubi, Cristina Mazzotti, Matteo Paganelli, Claudio Cerchione, Giovanni Marconi, Gerardo Musuraca, Giovanni Martinelli, Giorgia Simonetti. Targeting autophagy in acute lymphoblastic leukemia: Synergism between ponatinib, hydroxychloroquine and rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3001.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

Dear Editor, Acute myeloid leukemia (AML) patients carrying complex karyotype or aneuploidies have a very poor prognosis, with a 5-year overall survival (OS)

Simple Summary Gemtuzumab Ozogamicin (GO) is a drug approved for the treatment of acute myeloid leukemia (AML). It targets leukemic cells that express the CD33 molecule on their surface and brings the toxic agent calicheamicin inside the cell to kill it. Several studies have shown that AML patients can benefit of the addition of GO to chemotherapy during induction regimens, pre- and post-transplantation. Moreover, some disease features have been addressed or are under investigation for their capacity to predict response to GO, with the future aim of selecting AML patients that can mostly benefit of GO treatment. Abstract Acute myeloid leukemia (AML) is a complex hematological malignancy characterized by genetic and clinical heterogeneity and high mortality. Despite the recent introduction of novel pharmaceutical agents in hemato-oncology, few advancements have been made in AML for decades. In the last years, the therapeutic options have rapidly changed, with the approval of innovative compounds that provide new opportunities, together with new challenges for clinicians: among them, on 1 September, 2017 the Food and Drug Administration granted approval for Gemtuzumab Ozogamicin (GO) in combination with daunorubicin and cytarabine for the treatment of adult patients affected by newly diagnosed CD33+ AML. Benefits of GO-based regimens were also reported in the pre- and post-transplantation settings. Moreover, several biomarkers of GO response have been suggested, including expression of CD33 and multidrug resistance genes, cytogenetic and molecular profiles, minimal residual disease and stemness signatures. Among them, elevated CD33 expression on blast cells and non-adverse cytogenetic or molecular risk represent largely validated predictors of good response.

After being in the therapeutic wilderness for several decades, acute myeloid leukemia has been recently thrust into the limelight with a series of drug approvals. Technical refinements in production, genetic manipulation and chemical modification of monoclonal antibodies led to growing interest in antibodies-based treatment strategies. Much of the focus of these efforts in acute myeloid leukemia has been on CD33 as a target. On September 2, 2017, the U.S. Food and Drug Administration approved gemtuzumab ozogamicin for treatment of relapsed or refractory CD33sup+/supacute myeloid leukemia. This signals a new chapter in the long and unusual story of gemtuzumab ozogamicin, which was the first antibody-drug conjugate approved for human use by the Food and Drug Administration. In this review we have analyzed the history of this drug which, among several mishaps, is experiencing a second youth and still represents a field to be further explored.

Aneuploidy is a very rare and tissue-specific event in normal conditions, occurring in a low number of brain and liver cells. Its frequency increases in age-related disorders and is one of the hallmarks of cancer. Aneuploidy has been associated with defects in the spindle assembly checkpoint (SAC). However, the relationship between chromosome number alterations, SAC genes and tumor susceptibility remains unclear. Here, we provide a comprehensive review of SAC gene alterations at genomic and transcriptional level across human cancers and discuss the oncogenic and tumor suppressor functions of aneuploidy. SAC genes are rarely mutated but frequently overexpressed, with a negative prognostic impact on different tumor types. Both increased and decreased SAC gene expression show oncogenic potential in mice. SAC gene upregulation may drive aneuploidization and tumorigenesis through mitotic delay, coupled with additional oncogenic functions outside mitosis. The genomic background and environmental conditions influence the fate of aneuploid cells. Aneuploidy reduces cellular fitness. It induces growth and contact inhibition, mitotic and proteotoxic stress, cell senescence and production of reactive oxygen species. However, aneuploidy confers an evolutionary flexibility by favoring genome and chromosome instability (CIN), cellular adaptation, stem cell-like properties and immune escape. These properties represent the driving force of aneuploid cancers, especially under conditions of stress and pharmacological pressure, and are currently under investigation as potential therapeutic targets. Indeed, promising results have been obtained from synthetic lethal combinations exploiting CIN, mitotic defects, and aneuploidy-tolerating mechanisms as cancer vulnerability.

Background In Hodgkin Lymphoma (HL), neoplastic cells orchestrate an inflammatory microenvironment leading to sterile inflammation, T-cell anergy, and immune deficiency. Our group showed that in HL patients the aminoacid degrading enzyme Arginase-1 is increased, associated with poor outcome, and leads to arginine (Arg) deprivation. However, how the reduction of Arg in the extracellular milieu of the tumor microenvironment can contribute to neoplastic cell fitness is largely unknown. Aims To detect the adaptive response (via evaluation of global transcriptome and metabolome changes) in human HL cell lines exposed to Arg deprivation. Methods To better understand the impact of extra-cellular Arg1 deprivation on the metabolome of human cHL cells, four human cHL cell lines (L428, L540, HDMYZ and KM-H2) were individually cultured with customized complete media or lacking or Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments. After 48 hours of culture, the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by Illumina platform. Following normalization to DNA concentration, log transformation, and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups. Results While Arg deprivation did not affect cell viability but delayed cell cycle due to arrest in G2 phase in all tested cell lines, the effect of Arg deficiency on the cellular metabolome depended largely on the cell type examined with L428 and KMH2 cells having significantly changed metabolomes. Pyruvate was significantly higher in the KMH2 cells deprived of Arg compared to controls. Conversely, lactate was significantly lower, with increased levels of long-chain saturated fatty acids and long-chain polyunsaturated fatty acids (PUFAs). Taken together the metabolomics changes suggested that specific-amino acid deficiency can lead to an increase in free fatty acids synthases to preserve cytoplasmatic and mitochondrial membrane dynamics. Consistent with a metabolic rewiring to maintain mitochondrial integrity (the pyruvate is an important intermediary in the conversion of carbohydrates into fatty acids), the adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione in KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response (UPR, including XBP1, EIF2S1, EIF4A2, EIF4A3, ATF3, ATF4, DDIT4, EDEM1, GADD45B, SQSTM1, HMOX), NF-kB response to TNF (including RAF1, TNF, LIF, NKBIA, SGK1, BIRC3, ICAM1, BCL6, IL6, RELA, CDKN1A), p53 pathway and networks (including CDKN2B, STOM, TRAF4, RRAD, SESN1, FOXO3, SERPINB5, JAG2) and proteosome degradation (HSPA4, PSMD11, PSMD13, PSMD2, PSMA5, PSMA7, PSMC4), with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. All lines tested showed down-regulation of CCNI2, LCROL, MKI67, NCAPG, PEX10 and UFSP2, suggesting that early response to arginine deprivation includes modulation of UFMylation pathway, the most recently discovered post-translational protein modification system, whose biological function is largely unknown. Conclusions The removal of Arg from L428 and KMH2 resulted in changes in the specific-amino acid-related metabolites. The adaptive response to Arg-depleted environment increases oxidative stress and promotes a shift in glucose use in the attempt to preserve mitochondrial function. The cell-cycle arrest in G2, the increase of pyruvate availability and the upregulation of proteasome function via upregulation of the UFMylation pathway suggest the dependency of HL cell lines on mitochondrial function integrity. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Martinelli: Stemline Therapeutics: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Abbvie: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy. Di Raimondo: Pfizer: Honoraria; Jazz Pharmaceutical: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Background Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. Methods To understand the molecular bases of aneuploid acute myeloid leukemia (A‐AML), this study examined the genomic profile in 42 A‐AML cases and 35 euploid acute myeloid leukemia (E‐AML) cases. Results A‐AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E‐AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A‐AML, which was associated with a 3‐gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A‐AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E‐AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. Conclusions These findings indicate that aneuploidy‐related and leukemia‐specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets., Aneuploid acute myeloid leukemia (A‐AML) is associated with genomic and transcriptional alterations in the cell cycle and protein degradation pathways. The upregulation of PLK1 and CDC20 and the downregulation of RAD50 and of a p53‐related signature are hallmarks of A‐AML.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795∗; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that both affect the epigenetic regulation of DNA methylation. Although the proteins antagonistically regulate the epigenetic mark of 5-methylcytosine (5-mC), where DNMT3A catalyzes addition of 5-mC while TET2 oxidizes 5-mC as a first step in DNA demethylation, mutations in both genes appear in a similar spectrum of human hematopoietic malignancies. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases (OTKs) such as FLT3ITD, BCR-ABL1, JAK2V617F, MPLW515L or mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Moreover, while the mutations exert divergent effect on primitive hematopoietic progenitor cells, they lead to similar disease phenotypes, suggesting the roles of these mutations in hematopoietic malignancies may relate to mechanisms outside of DNA methylation. OTK-positive malignant cells accumulate high numbers of spontaneous and drug-induced DNA double-strand breaks (DSBs) in comparison to normal cells, but they manage to survive because of their enhanced/altered ability to repair these breaks. DSBs, the most lethal DNA lesions, are repaired by two major mechanisms, BRCA1/2-dependent homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ). Both HR and D-NHEJ repair DSBs in proliferating cells, while D-NHEJ plays a major role in quiescent cells. PARP1 -dependent alternative NHEJ (Alt-NHEJ) serves as back-up in both proliferating and quiescent cells. The existence of these redundant pathways creates the opportunity to employ a phenomenon called "synthetic lethality", which was originally applied to eliminate cancer cells with mutations in BRCA1 and BRCA2 by PARP inhibitor (PARPi). Our previous report [M. Nieborowska-Skorska et al., Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells. J Clin Invest 127, 2392-2406 (2017)] suggested that certain leukemias are sensitive to PARPi-triggered synthetic lethality. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing OTKs. Malignant TET2-deficient cells display downregulation of BRCA1 and LIG4 resulting in reduced activity of HR and D-NHEJ, respectively, and rely on Alt-NHEJ to protect them from the toxic effects of replication stress and drug-induced DSBs. Conversely, DNMT3A-deficient cells favor HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells are sensitive to PARPi treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity and/or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Our findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Tallman:Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties. Martinelli:Roche: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Daichii Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; AbbVie: Consultancy, Research Funding. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: 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, POLD1 and 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. Disclosures Martinelli: Roche: Consultancy; ARIAD: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy.

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.Leukemia advance online publication, 30 June 2017; doi:10.1038/leu.2017.183.

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 defines 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???=???0.002), ELN high risk (HR) (p?????0.001), lower white blood cell (WBC) count (p???=???0.040), TP53 loss, and/or mutations (p???

11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predictive of response and can help define therapeutic strategies. Our aim is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis by a SNP array-based genotyping approach. Methods: We performed SNP 6.0/Cytoscan HD (Affymetrix) on 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number (BioDiscovery) and R Core Team. Results: We found several genes preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p < .001) for genes mapping in these pathways: aberrant PD-1 signaling, loss of function of SMAD4 in cancer and SMAD4 MH2 Domain mutants in cancer. The pathways significantly (p < .001) deregulated in our cohort with single copy gain and homozygous amplification were: regulation of transcription and nucleic acid, negative regulation of metabolic processes, constitutive signaling by aberrant PI3K in cancer and PI3K/AKT network. In order to define driver alterations, we correlate deletions and losses with mutational data. We found losses are also targeted by mutations ( BRCA2, LRRK1), while deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations ( CASK deletion and MPP6 mutation, CDK6 deletion and PPM1D mutation, MAPT deletion and SPAG5mutation). Conclusions: We have identified new CNAs and pathways involving novel potential leukemia-related genes. The comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes in deregulated pathways deserve further investigation in order to identify new genes which could be relevant AML biomarkers. Ackn: ELN, AIL, AIRC,prog. Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY.

7038 Background: Nowadays, science is debating if autophagy in cancer can lead to therapy resistance or it can favor apoptosis. Autophagy pathways are involved pro-apoptotic mechanism, or they can improve stresses survival eliminating damaged mitochondria and proteins. Levels and activity of pro-apoptotic and anti-apoptotic proteins (eg. bcl-2 and p53), high levels of cAMP, and a pink/park complex could play as fulcrum on this lever. Our study aims to define the role of autophagy in AML. Methods: We analyzed 148 consecutive non M3 AML with Affymetrix SNP array. We screened all patients for TP53, FLT3, NMP1 mutations. Patients was treated with intensive induction chemotherapy regimens. Survival data were collected prospectively, with a median follow-up of 18 months. Results: Autophagy alteration (gene group 1: ULK1 CHR11; ULK1 CHR17; BECN1; ATG14; AMBRA1; UVRAG; ATG9A; ATG9B; PIK3C3; PIK3R4) was related to lower Complete Remission rate (CR%) after induction in univariate (p < .001) and multivariable regression model with age, karyotype, secondary AML, TP53 mutation (p = .014); autophagy alteration shown to confer worst Overal Survival (OS) (p < .001) and was significantly associated with complex karyotype and TP53 mutation (p < .001). We detected significant differences in term of survival independently both in gain and loss in group 1 genes (p < .001). Alterations in genes in cAMP pathway (group 2: SESN1; PRKAA1 CHR 3; PRKAB1: PRKAA1 CHR 1: PRKAG1 CHR11; PRKAG1 CHR 7; PRKAG3; PRKAB1) and in genes that could be related to a switch from a physiological role of autophagy to a resiliency mechanism (group 3: CCND1; BCL2; PINK1; PARK2; TP53; MDM1; MDM4) showed to confer worst OS (p < .001 in both groups); Alteration in group 2 and group 3 were related to lower CR% after induction (p < .001 in both groups). Whole Exome Sequencing on 56 patients in our set did not found any significant mutation in genes we analyzed with the exception of TP53. Conclusions: Alterations in autophagy regulator genes are associated with poor prognosis and therapy resistance. A loss in autophagy could block apoptosis, a gain could confer cell resiliency. Acknowledgements: ELN, AIL, AIRC, Progetto Regione-Università 2010-12,FP7 NGS-PTL, HARMONY

11611 Background: Necroptosis is a type of necrotic cell death involving several genes transcription and activation of molecular mechanisms as death receptors, interferon, toll-like receptors, intracellular RNA and DNA sensors.The process is leading by the family of receptor-interacting protein kinase ( RIPK3, RIPK2, RIPK1) and the MLKL substrate. Losses of RIPK3 or MLKL, as well as deficiency in apoptosis, could allow tumor cells to escape the immunomediated cells death (ICD). Methods: We performed SNP Arrays (Cytoscan HD and SNP 6.0, Affymetrix) on a cohort of 300 non-M3 AML patients at diagnosis and we analyzed the Overall Survival (OS) of our patients with deficiency on necroptosis pathways. Survival was analyzed with Kaplan-Mayer method and Log-Rank test. We further analyze the relevance of different prognostic factors by the use of COX-Hazard Ratio statistical analysis. Results: We find that 18 patients presented a loss of RIPK1 or MLKL (nobody presented losses in RIPK3/RIPK2) and 13/18 patients were older than 65 years old. The Overall Survival (OS) of patients with alterations in these genes is significantly lower than control group, with a median OS of 3 vs 6 month respectively (p

Background: The genetically heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL group that doesn’t have the most recurrent adult rearrangements (t(9;22); t(1;19); t(4;11)) is collectively referred to as “triple negative” (Ph-/-/-). CRLF2 is 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: Understand the genomic background of all Ph-/-/- ALL and subsequently clustering Ph-/-/- considering CRLF2 overexpression event, in order to define new biomarkers in these subgroups. Pts and Methods: Ph-/-/- pts were sequenced by WES (44pts/93 samples) and 92 B-Other pts were analyzed with NGS target seq (NTS) to validate the 8 most mutated genes (Fig1A). GEP were performed on 55 Ph-/-/-, 29 B-ALL Ph+ and on 7 donors. We cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in a 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 (Pancancer and/or RNASeq; dMLPA-MRC-Holland; SNP Array; 454 Junior and PCR). Results: WES analysis identified some recurrent mutated genes (NRAS, PAX5, KRAS, PTPN11, EP400, JAK2, TP53, CREBBP) previously reported to be involved in B-ALL, confirming the pivotal roles of these gene in ALL. For the first time we described a little known gene PKHD1L1 as highly mutated (7.2%). TP53 was the most mutated gene (Fig1A) and that between these gene is the only one associated to a worst OS (p=0.004). Combining our new Ph-/-/- GEP clustering, WES, NTS, Fusion and CNA results we identify a defined 2-clusters-subdivision (Gr1 and Gr2). The Gr2 (14.1% of all B-ALL) is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) co-overexpression. The Gr2 GEP is similar to Ph+ one (Fig1C). Gr1 represents 46.9% of all B-ALL. Fusion, CNA and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mut or del), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. We also validated not previously described fusions. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upreg in the group1 and CDK6 in the Gr2. Preliminary data seems to confirm an higher effect on cell viability of a TKI on Gr2 primary cell pt (vs Gr1 pt). Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B. This new subclassification identifies new potential therapeutic targets with available drugs (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; TKIs already effective on Ph+ and Ph-like) to test. Citation Format: Anna Ferrari, Silvia Vitali, Valentina Robustelli, Andrea Ghelli Luserna di Rorà, Eugenio Fonzi, Simona Righi, Carmen Baldazzi, Michela Tebaldi, Samanta Salvi, Cristina Papayannidis, Giovanni Marconi, Mariachiara Fontana, Enrica Imbrogno, Antonella Padella, Giorgia Simonetti, Alessandra Santoro, Jesus María Hernández-Rivas, Maria Teresa Bochicchio, Fabiana Mammoli, Benedetta Giannini, Nicoletta Testoni, Daniele Calistri, Massimiliano Bonafè, Gastone Castellani, Elena Sabattini, Daniel Remondini, Giovanni Martinelli. “3c-up” a new adult Philadelphia negative acute lymphoblastic leukemia subgroup: Novel molecular markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2140.

Introduction: PPM1D (wild-type p53-inducible protein phosphatase WIP1) is a member of the PP2C family serine/threonine phosphatase involved in negative regulation of cell stress response pathways, leading to suppression of p53 after stress. To restore p53 function through MDM2 inhibition (Nutlin-3a) is a promising approach in AML. Promising results of the combination of MDM2 inhibitors and WIP1 inhibitor (WIP1i), obtained in many preclinical studies of solid tumors, paved the way for their application in AML. We investigated whether the inhibition of WIP1 (GSK2830371) could sensitize AML cell lines and primary cells to Nutlin-3a (Nut-3a) in order to obtain a novel therapeutic strategy for AML patients, based on restoration of p53 activity. Methods: In vitro viability (by WST-1 reagent) and Annexin-V/PI apoptosis assay were performed on a panel of TP53-wt AML cells (MOLM-13, MV-4-11 and OCI-AML3), on TP53-mutated NOMO-1 AML cells and on primary AML samples. Gene expression profile (GEP) and Western Blot (WB) analyses were performed on MV-4-11 and NOMO-1 after 16h. Results: Combined inhibition of increasing dosage of Nut-3a (0.5 to 5 uM) and WIP1i (5 to 20 uM) synergistically reduces TP53-wt AML cells viability, while NOMO-1 resulted to be insensitive to the combination. The combination index analyses showed a synergistically effect of the combination of both compounds on TP53-wt AML cells. Annexin V/PI staining showed that WIP1i sensitizes TP53-wt AML cell lines and primary samples to Nut-3a-induced apoptosis, when compared with single treatment. No effect was seen in NOMO-1. GEP demonstrated that MV-4-11 cells exhibits a major response to drug combination with an upregulation of cell cycle control genes, a downregulation of DNA repair machinery genes, upregulation of MDM2 and of TP53-downstream genes (eg.CDKN1A), confirming the activation of p53 pathway. NOMO-1 cells showed upregulation of resiliency-mechanism and confirmed insensitivity to both treatment. WB analysis confirmed GEP data showing an increased expression of p53 and p21 in wt-TP53 cell line after 16h of combined-treatment. Conclusions: We identified a novel synergistic drug combination between Nut-3a and WIP1i that induces apoptosis in AML cell lines and primary samples. GEP and WB of TP53-wt MV-4-11 and TP53-mutated NOMO-1 cells showed mechanisms underlying drug sensitivity and resistance giving novel insights on potential markers of response and novel drugable targets. In vivo studies are needed to confirm these preclinical data. Supported by: AIRC, FP7-NGS-PTL, Fondazione del Monte, HARMONY. Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giovanni Marconi, Martina Pazzaglia, Matteo Bocconcelli, Antonella Padella, Simona Soverini, Ilaria Iacobucci, Cristina Papayannidis, Anna Ferrari, Maria Teresa Bochicchio, Enrica Imbrogno, Michele Cavo, Andrea Ghelli Luserna di Rora, Giorgia Simonetti, Giovanni Martinelli. Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2964.

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. To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. From March 2015 to December 2017, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Note: This abstract was not presented at the meeting. Citation Format: Giovanni Martinelli, Stefania Paolini, Claudio Cerchione, Alessandra Santoro, Valentina Robustelli, Simona Soverini, Caterina De Benedittis, Enrica Imbrogno, Andrea Ghelli Luserna Di Rora, Sarah Parisi, Chiara Sartor, Giovanni Marconi, Silvia Lo Monaco, Maria Chiara Abbenante, Maria Chiara Fontana, Antonella Padella, Anna Ferrari, Giorgia Simonetti, Elena Tenti, Federica Frabetti, Francesca Volpato, Samantha Bruno, Fabiana Mammoli, Maria Teresa Bocchicchio, Carmen Baldazzi, Cristina Papayannidis. Blinatumomab is safe and effective in relapsed and MRD positive B-ALL CD19+ patients: The bologna compassionate program experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3100.

Genomic and functional alterations of enzymatic activity drive cancer metabolic reprogramming along with mutations of tumor suppressors and oncogenes. IDH1/2 lesions represent a paradigmatic example in acute myeloid leukemia (AML). The study aimed to stratify AML patients based on their metabolic landscape and to map potential connections between their metabolic and genomic profile. The genomic landscape of 166 AML patients was obtained by whole exome sequencing. Variants were called by MuTect and Varscan2. Six additional cases were analyzed by targeted sequencing (SOPHiA GENETICS). Metabolites were quantified by mass spectrometry of bone marrow cells (35 CD34+ and 15 CD33+ AML from the above-mentioned cohort) and compared with CD34+ cord blood and CD33+ healthy blood cells (n=21 each, Metabolon) by Welch's t-test. In AML, 17% of somatic variants targeted metabolism-related genes: 38% were enzymes (according to Recon2), including 3% of electron transport chain genes encoded by mitochondrial DNA (COX1-3, ND1-6), 3% were AML-related genes with a known involvement in cell metabolism (e.g. IDH1/2, MYC, KRAS) and 59% were metabolic regulators, defined by gene ontology annotation. Ninety-one% of patients carried at least one mutation in a metabolism-related gene, with 43% of variants rated as damaging. The most represented pathways were lipid, carbohydrate, nucleotide (AK9, H6PD), amino acids (IDO2) and glucose metabolism (PKM, HK3). Principal component analysis of metabolic data showed a distinct profile between AML and healthy cells, with a predictive accuracy of 86% and 95% for CD34+ and CD33+ cells, respectively. Conversely, few differences were observed between CD34+ and CD33+ AML. Unsupervised hierarchical clustering clearly defined 3 AML clusters (C1-3). Moreover, 3 subgroups could be identified in C3 without ambiguous assignments. C1 was enriched for NPM1-mutated (mut) cases (83%, 33%, 27% in C1, C2 and C3, respectively, p=0.03). NPM1-mutated AML were distinguished by a 12-metabolites signature. They showed increased levels of spermidine, cytidine 2′ or 3′-monophosphate (P), thymidine 3′-monoP, uridine-2',3'-cyclic monoP and decrease of inosine 5'-monoP, suggesting altered polyamine, pyrimidine and purine metabolism. Moreover, NPM1-mut AML had reduced levels of intermediates involved in acyl carnitine, lysophospholipid, phosphatidylethanolamine and sphingolipid metabolism. Overall, mutations of metabolism-related genes are common in AML. We defined a metabolic-based classification of AML and identified a new metabolic signature based on 12 metabolites that distinguish NPM1-mut AML from wild-type cases and healthy CD34+/CD33+ cells. Major alterations in the nucleotide and polyamine pathways suggest novel potential therapeutic approaches. Supported by: EHA research fellowship award, AIRC, FP7-NGS-PTL, Fondazione del Monte. Citation Format: Giorgia Simonetti, Antonella Padella, Eugenio Fonzi, Martina Pazzaglia, Margherita Perricone, Maria Chiara Fontana, Samantha Bruno, Maria Teresa Bochicchio, Eugenia Franchini, Jacopo Nanni, Giovanni Marconi, Italo F. do Valle, Rossella De Tommaso, Anna Ferrari, Enrica Imbrogno, Claudio Cerchione, Cristina Papayannidis, Emanuela Ottaviani, Daniel Remondini, Giovanni Martinelli. Metabolic profiling defines a new characterization of acute myeloid leukemia and identifies NPM1-mutated cases as a distinct subgroup [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5279.