Breakpoint Cluster Research Articles

Impact of Simultaneous Presence of Multiple PML-RARA Isoforms on Phenotype in Patients with Acute Promyelocytic Leukaemia.

To determine the coexistence of multiple PML-RARA transcripts in adult APL (acute promyelotic leukaemia) patients, and its impact on the patients' laboratory parameters, treatment responses, and prognoses. Cross-sectional study. Place and Duration of the Study: Department of Medical Genetics, Medical Faculty of Necmettin Erbakan University, Konya, Turkiye, from January 2015 to March 2023. The study group consisted of individuals diagnosed with APL. RNA isolation was performed by taking blood or bone marrow samples and the presence of breakpoints in PML-RARA bcr1, bcr2, and bcr3 was detected using the real-time PCR. However, the quantification of PML-RARA fusion transcripts cannot be provided using the utilised kit. Twelve women and eight men were examined with a mean age of 38 years (range: 19-80), and 46.5 years (range: 22-60) were examined, respectively. When evaluating patients based on isoforms, it was found that 40% had multiple isoforms. Nineteen (95%) patients achieved haematologic remission after the treatment. Only one patient who had three different isoforms did not achieve remission. The estimated median survival for patients with a single isoform and those with multiple isoforms was 78.1 months (95% CI: 37.8-117.6) and 71.7 months (46.2-97.2), respectively. Two of the patients with multiple isoforms were lost in the early stage, whereas no early-stage mortality was recorded among patients with a single isoform. Identifying PML-RARA isoform subtypes is important for predicting prognosis and informing clinical follow-up. Acute promyelocytic leukaemia, Breakpoint cluster region, Isoform, PML-RARA.

Impact of Simultaneous Presence of Multiple PML-RARA Isoforms on Phenotype in Patients with Acute Promyelocytic Leukaemia.

To determine the coexistence of multiple PML-RARA transcripts in adult APL (acute promyelotic leukaemia) patients, and its impact on the patients' laboratory parameters, treatment responses, and prognoses. Cross-sectional study. Place and Duration of the Study: Department of Medical Genetics, Medical Faculty of Necmettin Erbakan University, Konya, Turkiye, from January 2015 to March 2023. The study group consisted of individuals diagnosed with APL. RNA isolation was performed by taking blood or bone marrow samples and the presence of breakpoints in PML-RARA bcr1, bcr2, and bcr3 was detected using the real-time PCR. However, the quantification of PML-RARA fusion transcripts cannot be provided using the utilised kit. Twelve women and eight men were examined with a mean age of 38 years (range: 19-80), and 46.5 years (range: 22-60) were examined, respectively. When evaluating patients based on isoforms, it was found that 40% had multiple isoforms. Nineteen (95%) patients achieved haematologic remission after the treatment. Only one patient who had three different isoforms did not achieve remission. The estimated median survival for patients with a single isoform and those with multiple isoforms was 78.1 months (95% CI: 37.8-117.6) and 71.7 months (46.2-97.2), respectively. Two of the patients with multiple isoforms were lost in the early stage, whereas no early-stage mortality was recorded among patients with a single isoform. Identifying PML-RARA isoform subtypes is important for predicting prognosis and informing clinical follow-up. Acute promyelocytic leukaemia, Breakpoint cluster region, Isoform, PML-RARA.

Correlation of T Regulatory Cells, Cytotoxic T-lymphocyte-associated Antigen 4, and Transforming Growth Factor-β1 with Treatment Response in Patients with Chronic Myeloid Leukemia Receiving Dasatinib Therapy.

Tyrosine kinase inhibitors improve chronic myeloid leukemia (CML) outcomes. Dasatinib inhibits breakpoint cluster region-Abelson 1 proto-oncogene tyrosine kinase better than imatinib in CML. T-regulatory cells prevent autoimmune diseases and aberrant immune responses by reducing oncoprotein antigen reactivity. They also reduce self-antigen-induced immune responses to maintain peripheral tolerance. In this study, T-regulatory cells in peripheral blood of chronic myeloid leukemia-chronic phase patients were measured, together with serum levels of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and transforming growth factor (TGF)-β1 at diagnosis and 3 months postdasatinib therapy. The Pathology and Clinical Haematology Departments at King George's Medical University, Lucknow, India, conducted this prospective analytical study. Forty CML-chronic patients and 10 healthy controls were analyzed. Flow cytometry was used to determine T-regulatory cell percentage in peripheral blood mononuclear cells of newly diagnosed CML patients before and after 3 months of dasatinib treatment; ELISA was used to measure serum levels of CTLA-4 and TGF-β1. T-regulatory cells, CTLA-4, and TGF-β1 significantly decreased in CML-chronic phase patients after 3 months of dasatinib therapy compared to the initial diagnosis. No significant change in T-regulatory cell, CTLA-4, or TGF-β1 percentages were seen between responders and poor responders. However, responders had a lower percentage of T-regulatory cells than suboptimal responders. The study concluded that dasatinib treatment improved response in CML patients with decreased Treg cells. Dasatinib reduces Treg-mediated immunological suppression, reducing CTLA-4 and TGF-β1 levels.

Leukocytosis and thrombocytosis after splenectomy: expected finding, infection, or something else: a case report

BackgroundLeukocytosis and thrombocytosis often follow splenectomy in blunt trauma patients, complicating the postoperative identification of infection. While the platelet count to white blood cell ratio provides diagnostic assistance to discern between expected laboratory alterations and infection, diagnoses such as leukemia are often overlooked.Case presentationA 53-year-old Hispanic male presented with abdominal pain, nausea, tachycardia, and focal peritonitis 4 days after being assaulted and struck multiple times in the abdomen. Initial white blood cell count was 38.4 × 109/L, platelet count was 691 × 109/L, and lipase was 55 U/L. Computed tomography abdomen/pelvis demonstrated a hematoma encasing the distal pancreas and abutting the stomach and colon. Emergent laparotomy revealed a nearly transected pancreas and devascularized colon, necessitating a distal pancreatectomy, splenectomy, and colonic resection with primary anastomosis. Postoperatively, he had a persistently elevated leukocytosis, thrombocytosis, segmented neutrophils, eosinophilia, and basophilia (peak at 70, 2293, 64, 1.1, and 1.2 × 109/L, respectively). Despite sepsis workup, including repeat computed tomography, no source was identified. Hematology/oncology was consulted for concern for hematologic etiology, with genetic testing and bone marrow biopsy performed. The diagnosis of breakpoint cluster–Abelson gene-positive chronic myeloid leukemia was made based on genetic tests, including polymerase chain reaction and fluorescence in situ hybridization analysis, which confirmed the presence of the Philadelphia chromosome. Bone marrow biopsy suggested a chronic phase. The patient was treated with hydroxyurea and transitioned to imatinib.ConclusionsThrombocytosis following splenectomy is a common complication and a plate count to white blood cell count ratio < 20 indicates infectious etiology. A significantly elevated white blood cell count (> 50 × 109/L) and thrombocytosis (> 2000 × 109/L) may suggest something more ominous, including chronic myeloid leukemia , particularly when elevated granulocyte counts are present. Chronic myeloid leukemia workup includes peripheral smear, bone marrow aspiration, and determination of Philadelphia chromosome. Post-splenectomy vaccines are still indicated within 14 days; however, the timing of immunization with cancer treatment must be considered. Tyrosine kinase inhibitors are the first-line therapy and benefits of pretreatment with hydroxyurea for cytoreduction remain under investigation. Additionally, tyrosine kinase inhibitors have been associated with gastrointestinal perforation and impaired wound healing, necessitating heightened attention in patients with a new bowel anastomosis.

Chronic myeloid leukemia: 2025 update on diagnosis, therapy, and monitoring.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm with an annual incidence of two cases/100 000. It accounts for approximately 15% of newly diagnosed cases of leukemia in adults. CML is characterized by a balanced genetic translocation, t(9;22) (q34;q11.2), involving a fusion of the Abelson murine leukemia (ABL1) gene from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. This rearrangement is known as the Philadelphia chromosome. The molecular consequence of this translocation is the generation of a BCR::ABL1 fusion oncogene, which in turn translates into a BCR::ABL1 oncoprotein. Four tyrosine kinase inhibitors (TKIs), imatinib, dasatinib, bosutinib, and nilotinib, are approved by the United States Food and Drug Administration (FDA) for first-line treatment of newly diagnosed CML in the chronic phase (CML-CP). Clinical trials with second and third-generation TKIs in frontline CML-CP therapy reported significantly deeper and faster responses but had no impact on survival prolongation, likely because of their potent efficacy and the availability of effective TKIs salvage therapies for patients who have a cytogenetic relapse with frontline TKI therapy. All four TKIs are equivalent if the aim of therapy is to improve survival. In younger patients with high-risk disease and in whom the aim of therapy is to induce a treatment-free remission status, second-generation TKIs may be favored. For CML post-failure on frontline therapy, second-line options include second and third-generation TKIs. Although potent and selective, these TKIs exhibit unique pharmacological profiles and response patterns relative to different patient and disease characteristics, such as patients' comorbidities and financial status, disease stage, and BCR::ABL1 mutational status. Patients who develop the T315I "gatekeeper" mutation display resistance to all currently available TKIs except ponatinib, asciminib, and olverembatinib. Allogeneic stem cell transplantation remains an important therapeutic option for patients with CML-CP and failure (due to resistance) of at least two TKIs and for all patients in advanced-phase disease. Older patients who have a cytogenetic relapse post-failure on all TKIs can maintain long-term survival if they continue a daily most effective/least toxic TKI, with or without the addition of non-TKI anti-CML agents (hydroxyurea, omacetaxine, azacitidine, decitabine, cytarabine, and others).

The Unusual Presentation of Bilateral Proptosis Presents a Dilemma in the Case of Juvenile Myelomonocytic Leukemia

AbstractJuvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative neoplasm. It is a rare pediatric neoplasm occurring in early childhood. Herein, we present a case of JMML in a 4-year-old girl admitted for primary complaints of protrusion of eyes and fever for the past 15 days not responding to any medications. With findings of splenomegaly and peripheral blood smear showing severe leukocytosis and increased monocytoid and blast cells, a myelomonocytic series neoplasm was suspected. Abelson (Abl) tyrosine kinase gene break point cluster (Bcr) gene (BCR-ABL1) was found to be negative on fluorescence in situ hybridization (FISH). Taking into consider all these findings, the patient was diagnosed with JMML. Since hematopoietic stem cell transplantation (HSCT) is not offered at our hospital, the patient was referred to another medical facility for the required procedure. This case highlights that bilateral proptosis could be a primary finding in early cases of JMML and should not be missed.

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development.

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.

Two recurrent types of IGH::5′ BCL2 breakpoints representing cytogenetic ins(14;18)(q32;q21q21) and t(14;18)(q32;q21), mediated by the VDJ and class switch recombination processes, respectively

We describe two types of IGH::BCL2 breakpoints involving the 5′ region of BCL2 (5′ BCL2). One was ins(14;18)(q32;q21q21) observed in 2 follicular lymphoma (FL) cases, in which IGH was cleaved at 3’ of IGHD and 5’ of IGHJ and BCL2 was cleaved at 5’ BCL2 and downstream regions, and a 281- or 201-kilobase pair fragment containing the BCL2 protein-coding sequences was invertedly inserted into IGH. In another type observed in 2 FL and 2 chronic lymphocytic leukemia (CLL) cases, breakage and reunion occurred within the switch region associated with IGHM (Sµ) and 5′ BCL2, creating IGH Sµ::5′ BCL2 fusion sequences on der(18)t(14;18)(q32;q21). The former is considered to be mediated by VDJ-recombination, while the latter by the class switch recombination process. There were no particular features in FL or CLL cases with IGH::5′ BCL2 breakpoints compared with those with t(14;18)(q32;q21)/IGH::BCL2 involving the 3′ breakpoint cluster regions.

Abstract 403: APOBEC3 enzymes induce chromosomal translocations in solid cancer

Abstract Introduction: Chromosomal translocations are common oncogenic drivers in human cancers, and an increasing number of translocations are being considered as crucial diagnostic and prognostic markers in clinics. In B-cell lymphoma, a number of chromosomal translocations are directed by the off-target activity of the activation-induced cytidine deaminase (AID; a B-cell specific apolipoprotein B mRNA-editing enzyme, catalytic polypeptode-like (APOBEC) family enzyme). However, in solid tumors, no such enzymes capable of directing chromosomal translocations have been identified. Given recent findings on APOBEC-mediated mutagenesis in solid cancers, APOBECs may be involved in the formation of chromosomal translocations in solid tumors, like AID does in B cells. In human, the APOBEC3 (A3) family consists of seven members (A3A-A3H) and is associated with C &amp;lt;t or="" c&amp;lt;g="" substitutions.="" recent="" evidence="" shows="" that="" a3a="" might="" be="" involved="" in="" chromosomal="" instability="" leading="" to="" drug="" resistance="" lung="" cancer.="" however,="" it="" has="" not="" been="" explored="" whether="" a3s="" generate="" dna="" dsbs,="" eventually="" translocations.="" this="" study,="" we="" investigated="" the="" role="" of="" and="" their="" mechanisms="" translocations="" solid="" cancer.Methods: We applied high-throughput genome-wide translocation sequencing (HTGTS) approach to map genome-wide chromosomal translocations induced by human A3s in mouse fibroblast and human epithelial cells. We correlated patterns of translocations induced by A3s with several other cellular features including transcription, DNA accessibility, DNA replication timing, and mutational signatures. We validated the contribution of A3 to the generation of oncogenic translocations in lung cancer. Results: Ectopic expression of A3s significantly increased chromosomal translocations in mouse fibroblasts and human epithelial cells. A3s and AID preferentially induced translocations in different parts of the genome, but some of the translocation hotspots were shared among them. Hierarchical clustering of hotspots showed that A3-mediated translocations were distinguished from AID-mediated translocations. Notably, the junction distribution of breakpoint clusters showed three distinct translocation patterns based on the locations of breakpoint clusters: (1) AID-preferred; (2) A3-preferred; and (3) AID/A3-mixed. AID-mediated translocations were heavily enriched near the transcription start site, whereas A3-mediated translocations were dispersed across the gene body and transcription end site. Furthermore, DNA replication timing and genomic architectures were linked to the formation of A3-mediated chromosomal translocations in solid cancer. Finally, A3 contributed to the formation of oncogenic rearrangements in lung cancer. Conclusions: A3 enzymes induced chromosomal translocations with distinct patterns compared to AID and facilitated chromosomal translocation formation in solid cancers. Citation Format: Taek-Chin Cheong, Qi Wang, Ahram Jang, Elif Karaca-Atabay, Roberto Chiarle. APOBEC3 enzymes induce chromosomal translocations in solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 403.

Novel secondary pyridinyl amides: Synthesis, in vitro antiproliferative screenings, and molecular docking studies

A novel series of secondary pyridinyl amides, reinforced with the methylsulfonyl pharmacophore unit, was designed and synthesized. Further, we determined the antiproliferative activities of these compounds through experimental assays and analyzed their interactions with Breakpoint Cluster Region/Abelson (bcr/abl), Epidermal Growth Factor Receptor (EGFR), and Human Epidermal Growth Factor Receptor 2 (HER2) tyrosine kinases using molecular autodocking techniques. We grouped these amides into three categories, classified by the type of spacer that binds the pyridine ring to the amide nitrogen atom. Additionally, within each group, we strategically positioned the amide moiety at the ortho, meta, or para position on the pyridyl ring. Among the studied amides, compound 3 exhibited the best overall antiproliferative activity against human immortalized myelogenous leukemia (K562) (IC50 = 26 µM), human leukemia monocytic cell line (THP-1) (IC50 = 81 µM), and human hepatocellular carcinoma (HepG2) (IC50 = 210 µM). Molecular docking studies revealed that compound 3 binds effectively to bcr/abl, EGFR, and HER2 tyrosine kinases, demonstrating strong interactions with crucial active site amino acids, indicative of its targeted antiproliferative mechanism. All investigated compounds showed either very minor or no cytotoxicity against human dermal fibroblast adult cells.

Computer-aided drug discovery of c-Abl kinase inhibitors from plant compounds against chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the neoplastic transformation of hematopoietic stem cells, driven by the Philadelphia (Ph) chromosome resulting from a translocation between chromosomes 9 and 22. This Ph chromosome harbors the breakpoint cluster region (BCR) and the Abelson (ABL) oncogene (BCR-ABL1) which have a constitutive tyrosine kinase activity. However, the tyrosine kinase activity of BCR-ABL1 have been identified as a key player in CML initiation and maintenance through c-Abl kinase. Despite advancements in tyrosine kinase inhibitors, challenges such as efficacy, safety concerns, and recurring drug resistance persist. This study aims to discover potential c-Abl kinase inhibitors from plant compounds with anti-leukemic properties, employing drug-likeness assessment, molecular docking, in silico pharmacokinetics (ADMET) screening, density function theory (DFT), and molecular dynamics simulations (MDS). Out of 58 screened compounds for drug-likeness, 44 were docked against c-Abl kinase. The top hit compound (isovitexin) and nilotinib (control drug) were subjected to rigorous analyses, including ADMET profiling, DFT evaluation, and MDS for 100 ns. Isovitexin demonstrated a notable binding affinity (-15.492 kcal/mol), closely comparable to nilotinib (-16.826 kcal/mol), showcasing a similar binding pose and superior structural stability and reactivity. While these findings suggest isovitexin as a potential c-Abl kinase inhibitor, further validation through urgent in vitro and in vivo experiments is imperative. This research holds promise for providing an alternative avenue to address existing CML treatment and management challenges. Communicated by Ramaswamy H. Sarma

Concomitant inhibition of the thioredoxin system and nonhomologous DNA repair potently sensitizes Philadelphia-positive lymphoid leukemia to tyrosine kinase inhibitors.

Breakpoint cluster region-Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients, 50% of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B-ALL cells, particularly in combination with other drugs. Here, we present that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib-treated, PRDX1-deficient cells revealed that the nonhomologous end-joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B-ALL cells. Accordingly, PRDX1-deficient Ph+ B-ALL cells were susceptible to NHEJ inhibitors. Finally, we demonstrated the potent efficacy of a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors against Ph+ B-ALL cell lines and primary cells, which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B-ALL.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

Abstract B015: Identification of novel natural compounds that induce DNA damage and chromosomal translocations

Abstract Background: Etoposide is a well-characterized chemotherapeutic that promotes DNA double-strand breaks (DSBs) and is associated with therapy-related chromosomal translocations and leukemias. Natural compounds including bioflavonoids, which are found in food as well as in dietary supplements, have been shown by our lab to induce DSBs and promote genome rearrangements. Given the potential for bioflavonoids to cause these harmful effects, we are investigating the potential for a wide array of other natural compounds and environmental agents to also induce DNA DSBs and chromosomal translocations. The purpose of this study was to perform a high throughput screen on a National Institutes of Health (NIH) library of natural compounds to determine their ability to cause DSBs and chromosomal translocations and implicate them as risk factors for leukemias. Methods: We obtained 2 libraires from NIH containing over 2000 natural compounds. Mouse embryonic stem cells (mES) were exposed to individual compounds at 50mM for one hour. Following exposure, DNA damage was measured through γ-H2AX immunocytochemistry and microscopy to quantify the level of DNA damage induced in comparison to etoposide. Compounds identified to cause greater than or equal to 20% of the damage induced by etoposide were prioritized for study of their potential to induce chromosomal translocations. We developed a mES GFP Reporter cell line with engineered exons of GFP contained within MLL and AF9 breakpoint cluster region inserts. Cells were exposed to individual compounds at 50mM for one hour. Following exposure, cells were screened over 7 days for the generation of chromosomal translocation events as measured by appearance of GFP+ cells. Results: Exposure to the NIH natural compound library has thus far generated 22 hits with 5 - 20% the amount of damage caused by etoposide and 29 hits with greater than or equal to 20% of the damage of etoposide out of the 1,200 compounds tested thus far. Top “hits” include known inhibitors of topoisomerase I and II. Other top “hits” include natural compounds curcumin, illudin, prodigiosin, and predorine. Testing of the ability of these “hits” to cause chromosomal translocations are still underway, but compounds, such as curcumin, are showing translocation GFP+ events. Conclusions: Data from this study can be used to inform the public and doctors of natural compounds that may be believed to be beneficial for human health but that can actually cause DNA damage and potential translocations. This data could generate potential drug candidates useful in the treatment of cancer. Etoposide is known to create DSBs but it also causes frequent translocation events. This study could identify compounds that are able to cause DNA DSBs, essential to the destruction of cancer cells, with a lower likelihood of causing chromosomal translocation events in healthy cells. Citation Format: Donna Goodenow, Henry Thompson, Elizabeth Toufekoulas, Heather Derby, Christine Richardson. Identification of novel natural compounds that induce DNA damage and chromosomal translocations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B015.

Sorafenib combination with imatinib enhances apoptotic and cell cycle arrest effects with no effect on viability and BCR-ABL expression of K-562 cells

Chronic myeloid leukemia (CML) is one of the myeloproliferative disorders that affect hematopoiesis. Historically, many therapies have been used for the management of this disease with moderate response. The discovery of tyrosine kinase inhibitors (TKIs), marked by the introduction of imatinib, revolutionized the therapy of CML. Resistance to imatinib, however, marks CML cells in refractory disease. Additionally, advanced stages of CML may still be refractory to available therapies. Sorafenib is an inhibitor of multiple intracellular and receptor protein kinases that is approved by the FDA for the treatment of advanced hepatocellular carcinoma patients and renal cell cancer. Other TKIs, such as erlotinib and sunitinib, are under study for the treatment of CML patients. In this study, the synergistic effect of the combination of imatinib and sorafenib on the K-562 CML cell line was evaluated. We used viability assay, cell cycle analysis, apoptosis assay, reverse transcription quantitative real-time PCR detect the expression of Breakpoint Cluster Region – abelson fusion oncogene and to evaluate the interaction of the combination. The study showed that the addition of sorafenib to imatinib did not produce a significant synergistic effect on the viability of cells. The combination, however, had a significant effect on cell cycle arrest and induction of apoptosis with no effect on the reduction of BCR-ABL mRNA expression on K-562 cells.

REAL LIFE DATA OF CHRONIC MYELOID LEUKEMIA PATIENTS IN ISPARTA

Objective&#x0D; Chronic myeloid leukemia is a myeloproliferative&#x0D; neoplasm with an incidence of 1–2 cases per 100&#x0D; 000 adults. Central to the pathogenesis of CML, is the&#x0D; fusion of the Abelson murine leukemia (ABL1) gene&#x0D; on chromosome 9 with the breakpoint cluster region&#x0D; (BCR) gene on chromosome 22. The therapeutic&#x0D; landscape changed dramatically with the development&#x0D; of the tyrosine kinase inhibitors (TKIs). This “targeted”&#x0D; approach altered the natural history of CML, improving&#x0D; the 10-year survival rate to 80-90%. This study aims to&#x0D; investigate the effective management of TKI treatment&#x0D; and overall survival in “real-life” CML patients and to&#x0D; discuss the results with current literature.&#x0D; Material and Method&#x0D; Fifty-eight patients who were diagnosed as CML&#x0D; between 2000 and 2018 in Suleyman Demirel&#x0D; University Hematology Department were evaluated.&#x0D; Patients’ clinical and laboratory characteristics, clinical&#x0D; and demographical features, treatment options, side&#x0D; effects and responses were evaluated in this study.&#x0D; Risk assessment and staging applied with World Health&#x0D; Organization criteria and Sokal Hasford and Eutos&#x0D; risk scoring system. We determined hematological,&#x0D; cytogenetic and molecular response according to&#x0D; European Leukemia Network criteria.&#x0D; Results&#x0D; The average age, male / female ratio and survival&#x0D; rates were found similar to those in literature. The&#x0D; age distribution (p = 0,001) and Charlson comorbidity&#x0D; index (p = 0.005) and Charlson comorbidity-age index&#x0D; (p = 0,000) had a statistically significant effect on&#x0D; overall survival. Age distribution (p = 0,029), Charlson&#x0D; comorbidity age index (p = 0,001) and major molecular&#x0D; response at 12 months (p = 0,028) were found to have&#x0D; a significant effect on disease-free survival. Major&#x0D; molecular response at 12 months (p = 0,006) also&#x0D; had a statistically significant effect on progression-free&#x0D; survival. Reticular fiber grade did not significantly affect&#x0D; overall survival, disease-free survival and progressionfree&#x0D; survival of patients.&#x0D; Conclusion&#x0D; These results suggest that CML is generally well&#x0D; managed with existing treatment options and that&#x0D; death occur more frequently due to other medical&#x0D; problems. In CML, Charlson indices have been shown&#x0D; to be significantly associated with overall survival and&#x0D; disease-free survival.

Chemoproteomics Reveals Glaucocalyxin A Induces Mitochondria-Dependent Apoptosis of Leukemia Cells via Covalently Binding to VDAC1.

Chronic myelogenous leukemia (CML) that is resistant to tyrosine kinase inhibitors is one of the deadliest hematologic malignancies, and the T315I mutation in the breakpoint cluster region-Abelson (BCR-ABL) kinase domain is the most prominent point mutation responsible for imatinib resistance in CML. Glaucocalyxin A (GLA), a natural bioactive product derived from the Rabdosia rubescens plant, has strong anticancer activity. In this study, the effect and molecular mechanism of GLA on imatinib-sensitive and imatinib-resistant CML cells harboring T315I mutation via a combined deconvolution strategy of chemoproteomics and label-free proteomics is investigated. The data demonstrated that GLA restrains proliferation and induces mitochondria-dependent apoptosis in both imatinib-sensitive and resistant CML cells. GLA covalently binds to the cysteine residues of mitochondrial voltage-dependent anion channels (VDACs), resulting in mitochondrial damage and overflow of intracellular apoptotic factors, eventually leading to apoptosis. In addition, the combination of GLA with elastin, a mitochondrial channel VDAC2/3 inhibitor, enhances mitochondria-dependent apoptosis in imatinib-sensitive and -resistant CML cells, representing a promising therapeutic approach for leukemia treatment. Taken together, the results show that GLA induces mitochondria-dependent apoptosis via covalently targeting VDACs in CML cells. GLA may thus be a candidate compound for the treatment of leukemia.

Distinct Pattern of ABL1 Genomic Breakpoints in Chronic Myeloid Leukemia and BCR::ABL1-Positive Acute Lymphoblastic Leukemia: Analysis of 884 Patients with Minor and Major BCR::ABL1 Fusion

The BCR::ABL1 fusion gene is a hallmark of chronic myeloid leukemia (CML) but is also found in patients with acute lymphoblastic leukemia (ALL). There are two main breakpoint clusters in BCR - “minor” (between exons 1-2, resulting in p190 fusion protein, prevalent in ALL and scarce in CML) and “Major” (between exons 13-15, resulting in p210 protein). On the ABL1 side, the breakpoints are mostly localized between exons 1-3. Due to large intronic areas where the breakpoints occur, only a few papers have been published focusing on the structure of BCR::ABL1 fusions. Moreover, with a single exception, these studies included only the Major- BCR::ABL1 patients. Here, we focused on the analysis of BCR::ABL1 breakpoints involving CML and ALL, children and adults, and Major and minor forms of fusion. To our knowledge, we present data on the largest cohort of patients with BCR::ABL1 fusion identified at the DNA level described to date. Genomic breakpoints were found in 884 patients with BCR::ABL1+ ALL (n=463) and CML (n=421) by multiplex long distance PCR/Sanger sequencing or by NGS Custom Target Enrichment. The RSS database, MEME software and RepeatMasker were used to search RSS, specific motifs known for mediation of DNA breaks (59 motifs) and interspersed and other types of repeats within particular DNA areas. The uniformity test performed on breakpoint distribution within the minor (n=356) and Major (n=528) BCR areas revealed a non-random pattern (p=2.34e-21 and p=2.56e-10, respectively) with breakpoints accumulated in the second halves of the intron 1 (minor BCR) and intron 13 (Major BCR). Within the ABL1 area, breakpoint sites were distributed more randomly (p=9.28e-03); however, the breakpoint distribution within the ABL1 differed significantly between patients with CML and ALL (p=7.43e-05; see Figure) with higher accumulation of breakpoints near the 5‘ end of the ABL1 intron 1 in CML and near the 3‘ end of the intron 1 in ALL. The difference was not driven by the type of fusion (minor vs. Major BCR) as it was still visible even if only Major BCR::ABL1+ patients were analysed (414 CML and 108 ALL; p=2.28e-03). Comparison between various groups of patients (females vs. males; adults vs. children; various age groups, minor vs. Major BCR fusion [in ALL patients]), did not reveal a significantly distinct breakpoint distribution in any BCR or ABL1 area. Analysis of genomic breakpoints showed that fusions are mostly formed in loci with short homologies (49%; 1-71bp, median=2bp), by blunt end junctions (36%) or by a junction with insertion of random nucleotides (13%; 1-42bp, median=3bp) strongly suggesting NHEJ mechanism of double-strand break repair in vast majority of cases. In 313 patients we compared BCR::ABL1 and the reciprocal ABL1::BCR fusion. Vast majority of the BCR::ABL1 and ABL1::BCR breakpoints (81% and 79% on BCR and ABL1 area, respectively) were located within ±100bp window, showing almost precise reciprocal translocation, with negligible losses/gains of DNA resulting from the fusion. Analysis of the association of breakpoints with DNA motifs and chromatin structure did not reveal any significant association with the localization of breakpoints. In conclusion, we show different breakpoint distribution in ABL1 between CML and BCR::ABL1+ ALL. The double-strand breaks are repaired by NHEJ; however, we have not found any DNA or epigenetic motif, which might be responsible for the breakpoints. Our data suggest that the differences in breakpoint distribution are due to different chromatin accessibility and/or different relative positions of the BCR and ABL1 genes throughout the cell development, reflecting the origin of CML in earlier stages of hematopoiesis compared to ALL. Supported by AZV (NU21-03-00128) and NICR No. LX22NPO5102.

Chromoanagenesis in Haematological Malignancy: Review of Samples from Patients with Acute Leukemia and MDS

Background: Complex chromosome rearrangements (CCRs) include a variety of structural aberrations grouped as chromothripsis, chromoanasynthesis and chromoplexy. Although the underlying mechanisms for these phenomena are unknown and likely to be different, they appear to originate from a single event. While chromothripsis and chromoanasynthesis affect limited genome sites their role and genome associations in haematological malignancy remain to be elucidated. Aims: This study aimed to provide clarity on the location, incidence and association with TP53 gene profile of chromothripsis (cth) and chromoanasynthesis (cha) events in MDS/AML. Methods: We analysed data from routine investigations carried out by haematological malignancy diagnostic service (HMDS) at University College London Hospital (UCLH) on 2,506 samples of acute myeloid leukaemia (AML) and myelodysplasia (MDS), including G-banding, FISH (Cytocell), chromosome microarray analysis (CMA, Agilent) and target NGS(Illumina). Analysis of 87 samples with complex bone marrow karyotypes were reported following the ISCN 2020 as: (i) cth(alternating disomy and heterozygous loss along a chromosome or chromosomal segment) or (ii) cha (deletions and one or two copy number gains in a single chromosome or chromosome region including copy number variants without the clustered breakpoints of chromothripsis). Statistical analysis used RStudio (v 1.4.1106) to carry Pearson's chi-squared (χ²) and Fisher's exact (2-sided) tests. Results: Complex karyotypes harbouring cth and cha type aberrations were detected in 65 out of 727 AML and 22 out of 1779 MDS samples. The cth events were seen alongside cha in 67% of cases, rarely presenting as a sole abnormality (2%), while cha alone was found in 31% of samples. Most frequently cth was mapped at chromosome 21 (17%), chromosome 7 (15%), chromosome 17 and 19 (13%) and chromosome 5 (11%) (Fig.1a). In this cohort of 87cases, chromothripsis was found to be associated with TP53 deletions ( TP53Del/WT) in 7 (7%) and with TP53 mutations ( TP53Dpl/mut) in 22 (25%), while concurrent TP53 deletions and mutations ( TP53Del/mut) were detected in 29 (33%) ( p = 0.0005). Samples with TP53Del/mut harboured cth in chromosomes 7 (7%), 17 (6%), 8, 9, 19 and 21 (5%) whilst cases with TP53Dpl/mut had cth in chromosomes 21 (9%), 19 (8%), 5 (7%) and 7 (5%). (Fig 1 b). The cha aberrations generally followed the genome location of cth events with some exception. Firstly, chromosomes X,13 and 21 appear to have only cth and is seen at slightly higher level on chromosome 19 (cth 13% vs cha 9%). Secondly, cha was most frequently seen on 5q (61 %), chromosome 17 (40%) and 7q (34%). However, the same chromosomes also had the greatest number of cases presenting with concurrent cth and cha, seen in 63/87 (72%) for chromosome 5, 46/87 (53%) for chromosome 17 and 43/87 (49%) for chromosome 7. Importantly, only cases harbouring 5q deletions showed a statistically significant association with TP53 status ( p = 0.0011) in this cohort irrespective of cth and cha presence. Notably, 24/29 cases (83%) with TP53Del/mut were cth positive and had 5q31 deletion. This finding is an important observation but not in keeping with reports of exclusive association of TP53 mutations with concurrent 5q and 7q loss (1, 2 and 3). The concurrent recording of cth and cha performed by this study could explain the discrepancy, most likely due to high frequency of cha events in 5q. Summary This study highlights issues pertinent to risk stratification in routine diagnostic settings: (i) MDS/AML cases with complex genome harbouring either or both cth and cha share common TP53 aberrations (del/mut) and (ii) deletion of 5q31 is positively associated with TP53 aberrations and presence of cth events. (iii) CMA offers a comprehensive, sensitive, fast and financially superior way to detect cth, cha events along with genome complexity compared to G banding complemented by FISH. Overall, combining CMA and TP53 mutation screening offers a fast, reliable way to detect high risk disease in MDS/AML. Keywords: Complex genome, CMA, chromothripsis, TP53 mutations