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.

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.

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.

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 <0.02). To further investigate the interplay between TP53 mutations and the HR pathway, we analysed the expression profiles of HR genes in 539 patients. We identified two groups of patients having higher (HR-high) or lower (HR-low) expression levels of HR genes. A panel of 5 genes was able to discriminate patients between the two groups (BRCA1, RAD54B, RMI2, UBE2T and XRCC2; AUC=0.9). Enrichment analysis on differentially expressed genes and gene set enrichment analysis showed that the cell cycle pathway, together with the G2/M transition/mitotic phase, E2F targets and the fatty acid metabolism pathways were upregulated in HR-high patients, while the pRB, EZH2, RPS14 and HOXA9 pathways were downregulated. Moreover, we observed that AML expressing CBFB-MYH1, RUNX1-RUNXT1 or carrying RAD21 mutations had higher chances to express lower levels of HR genes (HR-low), while patients with STAG2, SRSF2, U2AF1, FLT3-ITD alterations had higher chances of having higher expression of HR genes (p<0.05). NPM1-mutated cases without FLT3-ITD clustered within the HR-low profile (adj-p<0.05), while TP53 mutated cases tended to cluster in the HR-high group, although statistical significance was not reached. In conclusion, our data showed the presence of alterations in the DDR pathway that might be the reflection of driver events in AML. Functional studies will elucidate the functional impact of these alterations. The results suggested the presence of a therapeutic window that might be exploited with DDR inhibitors in molecularly-defined subgroups of patients. 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.