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High-risk B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive disease, often characterized by resistance to chemotherapy. A frequent feature of high-risk B-ALL is loss of function of the IKAROS (encoded by the IKZF1 gene) tumor suppressor. Here, we report that IKAROS regulates expression of the BCL2L1 gene (encodes the BCL-XL protein) in human B-ALL. Gain-of-function and loss-of-function experiments demonstrate that IKAROS binds to the BCL2L1 promoter, recruits histone deacetylase HDAC1, and represses BCL2L1 expression via chromatin remodeling. In leukemia, IKAROS’ function is impaired by oncogenic casein kinase II (CK2), which is overexpressed in B-ALL. Phosphorylation by CK2 reduces IKAROS binding and recruitment of HDAC1 to the BCL2L1 promoter. This results in a loss of IKAROS-mediated repression of BCL2L1 and increased expression of BCL-XL. Increased expression of BCL-XL and/or CK2, as well as reduced IKAROS expression, are associated with resistance to doxorubicin treatment. Molecular and pharmacological inhibition of CK2 with a specific inhibitor CX-4945, increases binding of IKAROS to the BCL2L1 promoter and enhances IKAROS-mediated repression of BCL2L1 in B-ALL. Treatment with CX-4945 increases sensitivity to doxorubicin in B-ALL, and reverses resistance to doxorubicin in multidrug-resistant B-ALL. Combination treatment with CX-4945 and doxorubicin show synergistic therapeutic effects in vitro and in preclinical models of high-risk B-ALL. Results reveal a novel signaling network that regulates chemoresistance in leukemia. These data lay the groundwork for clinical testing of a rationally designed, targeted therapy that combines the CK2 inhibitor, CX-4945, with doxorubicin for the treatment of hematopoietic malignancies.

BackgroundThe Hereditary Breast and Ovarian Cancer Syndrome (HBOC) occurs in families with a history of breast/ovarian cancer, presenting an autosomal dominant inheritance pattern.BRCA1andBRCA2are high penetrance genes associated with an increased risk of up to 20-fold for breast and ovarian cancer. However, only 20–30% of HBOC cases present pathogenic variants in those genes, and other DNA repair genes have emerged as increasing the risk for HBOC. In Brazil, variants inATM, ATR, CHEK2, MLH1, MSH2, MSH6, POLQ, PTEN,andTP53genes have been reported in up to 7.35% of the studied cases. Here we screened and characterized variants in 21 DNA repair genes in HBOC patients.MethodsWe systematically analyzed 708 amplicons encompassing the coding and flanking regions of 21 genes related to DNA repair pathways (ABRAXAS1,ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MRE11, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51, TP53andUIMC1). A total of 95 individuals with HBOC syndrome clinical suspicion in Southeast Brazil were sequenced, and 25 samples were evaluated for insertions/deletions inBRCA1/BRCA2genes. Identified variants were assessed in terms of population allele frequency and their functional effects were predicted through in silico algorithms.ResultsWe identified 80 variants in 19 genes. About 23.4% of the patients presented pathogenic variants inBRCA1, BRCA2andTP53, a frequency higher than that identified among previous studies in Brazil. We identified a novel variant inATR, which was predicted as pathogenic by in silico tools. The association analysis revealed 13 missense variants inABRAXAS1, BARD1, BRCA2, CHEK2, CDH1, MLH1, PALB2,andPMS2genes, as significantly associated with increased risk to HBOC, and the patients carrying those variants did not present large insertions or deletions inBRCA1/BRCA2genes.ConclusionsThis study embodies the third report of a multi-gene analysis in the Brazilian population, and addresses the first report of many germline variants associated with HBOC in Brazil. Although further functional analyses are necessary to better characterize the contribution of those variants to the phenotype, these findings would improve the risk estimation and clinical follow-up of patients with HBOC clinical suspicion.

The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz‐type2 (SPINT2/HAI‐2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI‐2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5‐Azacitidine (Aza), a DNA methyltransferase inhibitor. In MDS‐ and de novo AML‐BMMSC, Aza treatment resulted in a pronounced SPINT2/HAI‐2 levels up‐regulation. Moreover, Aza treatment of HD‐BMMSC did not improve SPINT2/HAI‐2 levels. To understand the role of SPINT2/HAI‐2 down‐regulation in BMMSC physiology, SPINT2/HAI‐2 expression was inhibited by lentivirus. SPINT2 underexpression resulted in an increased production of HGF by HS‐5 stromal cells and improved survival of CD34+ de novo AML cells. We also observed an increased adhesion of de novo AML hematopoietic cells to SPINT2/HAI‐2 silenced cells. Interestingly, BMMSC isolated from MDS and de novo AML patients had increased expression of the integrins CD49b, CD49d, and CD49e. Thus, SPINT2/HAI‐2 may contribute to functional and morphological abnormalities of the microenvironment niche and to stem/progenitor cancer cell progression. Hence, down‐regulation in SPINT2/HAI‐2 gene expression, due to methylation in MDS‐BMMSC and de novo AML‐BMMSC, provides novel insights into the pathogenic role of the leukemic bone marrow microenvironment.

The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL). The native E. colil-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP) in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase) was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance., Highlights • l-asparaginase is one of the first biologicals to be used and an essential drug for the treatment of acute lymphoblastic leukemia (ALL). • Current access to technology has facilitated the worldwide production of l-asparaginase and other biosimilars at lower prices. • Different quality control standards among countries pose a serious challenge to warrant quality, safety and efficacy of biosimilars. l-asparaginase is a cornerstone drug in the treatment of acute lymphoblastic leukemia (ALL). A cheaper l-asparaginase biosimilar was introduced in the Brazilian market in 2017. This report describes the bioanalytical evaluation of such biosimilar, which revealed the presence of at least 12 different contaminating host-cell proteins (HCP), that accounted for 19 to 37% of the peptides identified by three independent mass spectrometry analyses. The second most abundant HCP alone was confirmed by an orthogonal method to represent 2.4% of the total protein mass. Studies carried out in mice showed lower plasma bioavailability and higher anti-asparaginase antibodies titres for the biosimilar in comparison to the well-known Medac l-asparaginase previously used in the Country.

Despite the success achieved in the treatment of acute lymphoblastic leukemia (ALL), the search for new drugs featuring selectivity against leukemia cells and effectiveness to prevent relapsed ALL is still highly desirable. Here, we described the synthesis of several novel 3-substituted and 3,6-disubstituted-2-carboalkoxy indoles followed by the elucidation of their mechanism of action and in vivo anti-leukemia efficacy. The synthesis of 3-substituted-2-carboalkoxy indoles relied on two Heck arylations of methyl acrylate and methyl cinnamates respectively, to generate β,β-disubstituted acrylates followed by an efficient Cadogan-Sundberg reaction of these latter intermediates. The method developed led to the synthesis of twenty-one novel functionalized indoles. Of these, indole 20 showed selective cytotoxicity against leukemia cells at the nanomolar scale, and, therefore, it was selected for the investigation of its mechanism of action. Indole 20 was found to target tubulin leading to G2/M cell cycle arrest, DNA damage and apoptosis. Indole 20 decreased β-tubulin protein in leukemia cells in a time-dependent manner and induced depolymerization of the microtubule network in Hela cells, thus fully characterizing its microtubule destabilizer activity. The connectivity map analysis of HL60 promyelocytic leukemia cells treated with indole 20 revealed a transcriptional profile similar to that of cells treated with prostaglandins, apparently due to the induction of cellular differentiation as addressed by the expression of CD11 and CD14 markers. Finally, indole 20 given intraperitoneally, at 10 mg/kg, 5x/week significantly prolonged the overall survival of NOD/SCID mice transplanted with RS4; 11 B-ALL cells.

Summary Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells., Graphical Abstract, Highlights • 3,4,5-trimethoxy-N-acylhydrazones bind to the colchicine site of tubulin • 12 forms a single H-bond with αThr179 and causes steric hindrance of tubulin βT7 loop • 3,4,5-trimethoxy-N-acylhydrazones feature low toxicity • 12 shows therapeutic effect against multidrug-resistant acute lymphoblastic leukemia, Drugs; Biological Sciences; Molecular Biology; Structural Biology; Cancer

Acute leukemias are a heterogeneous group of aggressive malignant neoplasms associated with severe morbidities due to the nonselectivity of current chemotherapeutic drugs to nonmalignant cells. The investigation of novel natural and synthetic structures that might be used for the development of new drugs with greater efficiency and selectivity to leukemic cells is mandatory. In this context, thiosemicarbazones have been well described in the literature by their several biological properties and their reaction is known as versatile, low-cost, and highly chemoselective. With this perspective, this study aimed to investigate the cytotoxic effect and the main death mechanisms of a novel thiosemicarbazone (LAP17) on acute leukemia cell lines K562 and Jurkat. The results show that the strong cytotoxic effect of LAP17 to leukemic cells is due to apoptosis induction, which resulted in caspase-3 activation and DNA fragmentation. Intrinsic apoptosis seems to be related to the inversion of Bax/Bcl-2 expression, ΔΨm loss, and AIF release, whereas extrinsic apoptosis was initiated by FasR. Gene-expression profiling of HL-60 cells treated with LAP17 by the microarray technique revealed a significant enrichment of gene sets related to cell cycle arrest at G2/M. Accordingly, K562 and Jurkat cells treated with LAP17 revealed a clear arrest at G2/M phase. Taking into consideration that LAP17 was not cytotoxic to nonhematological cells (peripheral blood mononuclear cell and erythrocytes), these results suggest that LAP17 is a promising new compound that might be used as a prototype for the development of new antileukemic agents.

Anacardium occidentale leaves are used in folk medicine due its therapeutic properties attributed to phenolic compounds. Therefore, this study was undertaken on its hydroethanolic leaf extract (AoHE) to evaluate cytotoxicity and apoptosis induction on acute lymphoblastic leukaemia cells. Results indicated that AoHE interfered in the cell cycle progression, inducing apoptosis by activation of casp3 at lower concentrations, thence, a promising candidate for the development of new cancer drugs.

3-(Thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboximidamides were efficiently prepared through a cyclocondensation of thiophenylchalcones with aminoguanidine hydrochloride under ultrasonic conditions in the presence of KOH and ethanol as a green solvent in short reaction times (15-35 min) and good yields (62-95%). All compounds produced were evaluated against the human Jurkat and RS4;11 acute lymphoblastic leukemia cell lines of T- and B-cell origin, respectively, and the K562 myelogenous leukemia cell line. Six compounds presented half maximal inhibitory concentration (IC50) values around 15 µmol L-1 and five compounds presented IC50 values around 40 µmol L-1 for at least one of the three cell lines analyzed. One compound was not significantly cytotoxic, presenting IC50 value > 100 µmol L-1.

CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL. Citation Format: Soumya C. Iyer, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jon Payne, Pavan Kumar Dhanyam Raju, Bihua Tan, Mary McGrath, Yevgeniya Bamme, Mario Solimon, Nathalia Moreno Cury, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [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 871.

Objective The aim of this study was to investigate whether the Ala55Val and -866G>A polymorphisms of the UCP2 gene are related to weight loss and changes in body composition after bariatric surgery performed by Roux-en-Y gastric bypass (RYGB). Methods This longitudinal study enrolled obese patients submitted to RYGB. Data regarding weight (kg), body mass index (kg/m2), fat-free mass (FFM; kg), fat mass (kg), weight loss (kg and %), and percent excess weight loss were collected from both preoperative and 1-y postoperative medical records. Polymorphisms were genotyped by allelic discrimination using real-time polymerase chain reaction and TaqMan-predesigned single nucleotide polymorphism Genotyping Assay kits (Applied Biosystems, Foster City, CA, USA). The t test was used to compare variables between genotypes of each polymorphism to analyze the dominant and recessive models. Linear regression models were used to adjust the effects of initial weight, age, and sex on the variation of weight and body composition (P Results We analyzed 150 severely obese individuals (age 47.2 ± 10.5 y; 80% women). Genotype analysis showed a greater prevalence of heterozygous GA (41.3%) for -866G>A polymorphism and CT (39.3%) for Ala55Val polymorphism. Individuals who carried the T (CT+TT) and A (GA+AA) mutated alleles for Ala55Val and -866G>A, respectively, showed a higher weight and FFM loss. Conclusion The mutated alleles T for Ala55Val and A for -866G>A polymorphism could be biomarkers of weight loss 1 y after RYGB.

In recent years, the role of tumor microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stem/stromal cell (BMMSC) contribution to disease progression remains poorly explored. We had previously performed a microarray analysis of myelodysplastic syndrome (MDS) patient-derived BMMSC (MDS-BMMSC) and found an underexpression of HAI-2/SPINT2, an endogenous inhibitor of the hepatocyte growth factor (HGF) activator. This gene has been described as methylated in various cancer types and has been associated with disease progression. Despite of being related to the pathogenesis of several neoplasms, the role of HAI-2/SPINT2 has not yet been fully elucidated in hematological diseases, such as MDS and acute myeloid leukemia (AML). Thus, the aim of this study was to evaluate HAI-2/SPINT2 expression in derived BMMSC and total bone marrow (BM) of healthy donors (HD), MDS and AML patients as well as in BMMSC treated with 5-Azacitidine (Aza), a DNA methyltransferase (DNMT) inhibitor. To achieve this, we collected BM hematopoietic cells and plastic-adherent BMMSC from aspirates of HD, MDS and AML patients. BMMSC were expanded to passage 4 and defined as CD73+/CD90+/CD105+/CD45-/CD34-/CD31-/HLA-DR-. A total of 29 HD and 121 patients at diagnosis (MDS=72 [low-risk=46, high-risk=26], AML with myelodysplastic related changes (AML-MRC)=17 and de novo AML=32) were included. HAI-2/SPINT2 mRNA was significantly decreased in MDS- (0.34[0.01-2.06];P Disclosures No relevant conflicts of interest to declare.