ALBERT

Description: The well-established association between TP53 mutations and adverse clinical outcome in a range of human cancers reflects the importance of p53 protein in regulating tumor-cell growth and survival. Although it is theoretically possible for p53 dysfunction to arise through mechanisms that do not involve TP53 mutation, such a phenomenon has not previously been demonstrated in a sporadic tumor. Here, we show that p53 dysfunction in B-cell chronic lymphocytic leukemia (CLL) can occur in the absence of TP53 mutation and that such dysfunction is associated with mutation of the gene encoding ATM, a kinase implicated in p53 activation. Forty-three patients with CLL were examined for p53 dysfunction, as detected by impaired up-regulation of p53 and of the p53-dependent protein p21CIP1/WAF1 after exposure to ionizing radiation (IR). Thirty (70%) patients had normal p53 responses and underwent progressive IR-induced apoptosis. In 13 (30%) patients, p21 up-regulation was markedly impaired, indicating p53 dysfunction. Six (14%) of these patients with p53 dysfunction had increased baseline levels of p53, were found to have TP53 mutations, and were completely resistant to IR-induced apoptosis. In the other 7 (16%) patients with p53 dysfunction, IR-induced p53 up-regulation and apoptosis were markedly impaired, but baseline levels of p53 were not increased, and no TP53 mutations were detected. Each of these patients was found to have at least one ATM mutation, and a variable reduction in ATM protein was detected in all 4 patients examined. This is the first study to provide a direct demonstration that p53 dysfunction can arise in a sporadic tumor by a mechanism that does not involve TP53 mutation.

Description: Abstract 3439 Poster Board III-327 Chronic lymphocytic leukaemia (CLL) is a malignancy with a variable clinical course in which a proportion of patients exhibits rapid clinical progression despite treatment. One of the major causes of treatment resistance is alterations in the ATM/p53 pathway imposed by mutations in either the ATM or TP53 genes. Consequently, there is an urgent need to devise novel therapeutic approaches that will be able to counteract the p53 apoptotic defect in these tumours. We have previously shown that DNA damage induces a complex ATM-dependent network of pro-survival and pro-apoptotic transcriptional responses (both p53-dependent and -independent) and that the balance between these responses determines CLL cellular death. Therefore, it is plausible to expect that manipulation of ATM-dependent transcription to either reduce pro-survival or increase pro-apoptotic signals can sensitise ATM and TP53 mutant CLL tumours to DNA damaging agents. Individual transcription factors (TFs) that govern ATM-dependent transcription are largely unknown. In this study we aimed to identify those factors by employing a DNA/Protein Transcription Factor ComboArray (Panomics/Affymetrix) which includes 345 DNA binding motifs for a range of transcription factors, DNA binding proteins and response elements. We compared the ability of nuclear cell extracts from 3 combined ATM wildtype primary CLL samples and 3 combined ATM mutant primary CLL samples to bind to biotin-labelled DNA binding motifs prior to irradiation (IR)-induced DNA damage, 2h and 6h post-IR. Following hybridisation of nuclear protein-bound biotin-labelled probes to the array and HRP visualisation, we identified 49 binding motifs (several of which were detected more than once through alternative sequences) which, in response to DNA damage, exhibited reduced binding in ATM mutant compared to the ATM wildtype CLL nuclear extracts. The most prominent differentially bound DNA binding motifs included those for GATA1 and 2, Transcriptional enhancer factor 1 (TEF1), c-Rel, Aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator binding element (AhR/Arnt), forkhead box I1 (HFH-3), Slow/Cardiac Troponin C (cTnC/CEF-2), E2A immunoglobulin enhancer binding factors (E12/E47), Pax-4, Wilms tumour 1 (WT1), antioxidant recognition element (ARE) and interferon-a stimulated response element (ISRE). We validated differential binding of individual TFs by electro-mobility shift analysis (EMSA) and selected six that were positively corroborated in an independent cohort of primary ATM mutant and ATM wildtype CLL tumour cells. We subsequently investigated the impact of altering the activity of the identified ATM-dependent TFs on the sensitivity of ATM mutant CLL tumours to DNA damage. Among the selected TFs, as a proof of principle, ARE demonstrated both ATM-dependent binding by EMSA as well as the capacity to modulate the DNA damage response in CLL cells: pharmacological activation of this TF by Dimethyl fumarate (DMF) sensitised ATM mutant cells to IR-induced DNA damage. In summary, we have identified a number of ATM-regulated transcription factors that could be directly or indirectly targeted to increase the sensitivity of CLL cells with a defective ATM/p53 pathway to DNA damaging agents. We also suggest that the DNA damage-dependent TF screen represents a feasible approach to identify novel molecular targets that may sensitise other subtypes of treatment-resistant CLL tumours. Disclosures No relevant conflicts of interest to declare.

Description: Recent advances in whole genome assessment have highlighted the presence of marked intraclonal heterogeneity in chronic lymphocytic leukaemia (CLL). It has been suggested that this heterogeneity plays an important role in disease progression and that treatment may facilitate the outgrowth of CLL subclones with a high proliferative advantage. Recent years have also seen the development of a number of novel therapeutic approaches for CLL, including a range of targeted treatments as well as immunochemotherapy. Consequently, there is a need to assess the effects that various treatments have on the subclonal architecture of CLL during disease progression as any changes could potentially influence future clinical decisions. In this study a cohort of 133 CLL tumours was analysed for the mutational status of 6 genes recurrently mutated in CLL; ATM, TP53, SF3B1, MYD88, BIRC3, and NOTCH1; by deep targeted sequencing with an allele depth of 3000-5000 reads. Mutation(s) of at least one gene was detected in 74 of these patients. Prior to treatment, mutations with allelic frequencies (AF) both greater than and less than 50% were identified for all genes. However, the dominant mutation of ATM, TP53, BIRC3 or NOTCH1, presented with an AF greater than 50% in the majority of cases, whereas SF3B1 and MYD88 mutations typically exhibited an AF less than 50%. Generally, there was frequent coincidence of ATM mutations with SF3B1 or BIRC3 mutations; NOTCH1 mutations often occurred concurrently with either ATM, BIRC3 or SF3B1. Occasionally, TP53 mutations were found concommitantly with SF3B1 mutations and less frequently with BIRC3 mutations, whereas sole MYD88 or TP53 mutations characterised another two subsets of patients. Additionally, BIRC3, SF3B1 and MyD88 mutations were mutually exclusive in this cohort, as were ATM and TP53 mutations, with the exception of 2 cases upon relapse. Time to first treatment (TTFT) and overall survival (OS) were shorter for patients with one or more mutated gene than those without a mutation in any of these six genes. Furthermore, the presence of mutations either in multiple genes or multiple mutations within a gene conferred a more inferior TTFT. To determine the impact of therapy upon the mutational status, 31 paired pre-treatment and relapse samples were assessed. The response to treatment was heterogeneous with AFs of mutations of all the genes both increasing and decreasing in various patients irrespective of the treatment regimen. The most frequent response observed in this cohort of patients was the appearance of novel, sometimes multiple mutations of TP53 which were undetected in pre-treatment samples. Similarly, we observed the emergence of one or more novel ATM and NOTCH1 mutations and less frequently BIRC3 and SF3B1 upon relapse in a subset of patients. Of note, changes in mutational status did not appear to be influenced by the type of treatment, chemotherapy (n=19) vs chemoimmunotherapy (n=12). Patient follow-up cannot determine whether changes in subclonal architecture are treatment-induced or a consequence of natural disease progression, thus we monitored the CLL subclonal architecture of 5 representative CLL cases during xenotransplantation by multiplexed-FISH analysis. In the absence of treatment, 3 of the 5 CLL xenografts faithfully recapitulated the subclonal architecture of the patient samples. Intriguingly however, in the remaining 2 untreated xenografts the subclonal architecture significantly deviated (P

Description: PALB2 protein is one of the principal components of Homologous Recombination Repair (HRR) that facilitates recruitment of other HRR proteins such as BRCA1, BRCA2 and Rad51. Biallelic PALB2 inactivation is associated with a severe form of Fanconi Anemia (FA-N), however, biallelic PALB2 mutations also occur in patients with a FA-N syndrome variant characterised by a predisposition to B-cell lymphoma development. The association of PALB2 with FA-N and FA-N variant suggests that PALB2 may play a pathogenic role in a wider range of sporadic haematopoietic malignancies. To address this possibility, the mutational status of the PALB2 gene was assessed in 30 myelodysplastic syndrome (MDS) samples, 23 acute lymphoblastic leukaemias (ALL), 171 chronic lymphocytic leukaemias (CLL) and 24 paediatric non-Hodgkin lymphomas (NHL) of T- or B-cell origin. Overall, pathogenic (truncating or splicing errors)orlikely pathogenic sequence changes (missense alterations not previously reported but predicted to alter protein function) were detected in 17 (6.7%) of these patients. PALB2 was most frequently altered in NHL (25%), followed by MDS (6%), CLL (5%) and ALL (4%). The overall incidence of changes observed for B-cell NHL was higher than the frequency detected in T-cell NHL. PALB2 sequence changes were distributed across the whole gene including the DNA binding, BRCA1-interacting and BRCA2-interacting domains. Of note, a single truncating mutation; in the BRCA1-interacting domain, c.886delA; was detected in three cases, two with progressive CLL and one with MDS and interestingly only became detectable in one of these CLL patients following ibrutinib treatment. Furthermore, a polymorphic variant (c.2993G〉A), previously associated with an increased breast cancer risk, was present at higher frequency than in the general population in cohorts of both CLL and ALL patients. HRR defective tumour cells can be targeted by pharmacological inhibition of co-operating DNA repair pathways using the principal of synthetic lethality. To explore whether this concept can be utilised in the context of PALB2 deficiency, we evaluated the effectiveness of inhibition of two pathways; PARP and ATR; that co-operate with PALB2. The cytotoxicity induced by either an ATR inhibitor or a PARP inhibitor was assessed in lymphoblastoid cell lines derived from a paediatric B-cell NHL patient carrying the c.1676_1677delAAinsG and c.2586+1G〉A mutations. As predicted, exposure of lymphoid cells with PALB2 mutations to either ATR or PARP inhibitor alone exerted a cytotoxic effect which was enhanced when both inhibitors were applied in combination. In conclusion, our results suggest that PALB2 is altered in a subset of patients with different haematopoietic malignancies. This potentially provides another avenue for targeted therapies utilising the concept of synthetic lethality via application of PARP and ATR inhibitors that are currently being tested in clinical trials. Disclosures Janic: Novo Nordisk: Other: Paid Instructor, Research Funding; Baxter: Other: Paid Instructor, Research Funding; Bayer: Other: Paid Instructor, Research Funding; Pfizer: Other: Paid Instructor, Research Funding; Octopharma: Research Funding.

Description: Abstract 467FN2 The Ataxia Telangiectasia Mutated (ATM) gene located at 11q23 plays a central role in DNA damage response. Deletion of 11q23 occurs in approximately 20–30% chronic lymphocytic leukemia (CLL) cases and in 30% of these exhibit mutations in their remaining ATM allele. A smaller proportion of CLL tumours exhibit the presence of ATM mutations in the absence of 11q deletion. We have previously shown that ATM mutations are associated with a shorter overall survival from diagnosis and treatment free survival (TFS) in an unselected CLL cohort. Furthermore, several prospective and retrospective studies, including the UKCLL4 trial, show an inferior overall survival and progression free survival after therapy (OS and PFS) in cases with an 11q deletion treated with alkylating agents and/or purine analogues. The entire ATM coding region was screened for sequence changes using high-performance liquid chromatography and sequencing and known polymorphisms excluded. Screening was performed on 238/777 cases enrolled in the UKCLL4 trial, including all available cases with 11q deletions, with the remaining cases being randomly selected. Other than significant enrichment for patients with 11q deletion (p

Description: Polycomb group (PcG) proteins are chromatin modifiers that are necessary for the maintenance and renewal of embryonic and adult stem cells. However, overexpression of the PcG protein, Bmi-1, causes lymphoma in transgenic mice. We show that Bmi-1 is up-regulated in Hodgkin lymphoma (HL) cells by the Epstein-Barr virus (EBV) oncogene latent membrane protein-1 (LMP1) and that this up-regulation is mediated by NF-κB signaling. We also show that Bmi-1 is up-regulated by NF-κB in EBV-negative HL cells. Down-regulation of LMP1 and Bmi-1 decreased the survival of HL cells, suggesting that Bmi-1 may mediate the prosurvival effects of LMP1-induced NF-κB signaling in HL cells. Transcriptional targets of Bmi-1 were identified after its knockdown in an HL cell line. We show here that Bmi-1 and LMP1 down-regulate the ataxia telangiectasia–mutated (ATM) tumor suppressor and conclude that Bmi-1 contributes to LMP1-induced oncogenesis in HL.

Description: Current prognostic indicators such as age, sex, and white blood cell count (WBC) fail to identify all children with more aggressive forms of B-precursor acute lymphoblastic leukemia (ALL), and a proportion of patients without poor prognostic indicators still relapse. Results obtained from an analysis of 65 pediatic B-precursor ALL patients indicated that subclone formation leading to clonal diversity, as detected by Ig and T-cell receptor (TCR) gene rearrangements, may represent a very useful prognostic indicator, independent of age, sex, and WBC. Disease-free survival was significantly shorter in those patients showing clonal diversity at presentation. Furthermore, clonal diversity was detected not only in the majority of high-risk patients who relapsed but was also associated with a high probability of relapse in standard-risk patients. Sixty-five percent (13/20) of standard-risk patients who also showed clonal diversity subsequently relapsed, whereas the percentage of relapses among standard-risk patients without clonal diversity was much lower at 19% (7/36). Continued clonal evolution during disease progression is an important feature of aggressive B-precursor ALL. All 5 patients with clonal diversity who were followed up in our study showed a change in the pattern of clonality between presentation and relapse. This implies an important role for clonal diversity as a mechanism of disease progression through the process of clonal variation and clonal selection. © 1998 by The American Society of Hematology.

Description: The ataxia telangiectasia mutated (ATM) protein is the principal activator of the p53 protein in the response to DNA double-strand breaks. Mutations in the ATM gene have been previously found in B-cell chronic lymphocytic leukemias (B-CLLs) but their clinical significance is unknown. We analyzed 155 CLL tumors and found 12% with ATM mutations and 4% with TP53 mutations; 2 tumors contained mutations in both genes. Retrospective analysis on selected samples indicated that the ATM mutations were usually present at diagnosis. Compared with patients with wild-type ATM/TP53 genes, patients with ATM mutations had statistically significantly reduced overall and treatment-free survival. Although present in both IGVH mutation subgroups, ATM mutations were associated with unmutated IGVH genes and they provided independent prognostic information on multivariate analysis. Mutations in the ATM gene resulted in impaired in vitro DNA damage responses. Tumors with ATM mutations only partially correlated with tumors with loss of an ATM allele through an 11q deletion and, interestingly, those 11q-deleted tumors with a second wild-type ATM allele had a preserved DNA damage response. The majority of patients with ATM mutations were refractory to DNA damaging chemotherapeutic drugs and as such might benefit from therapies that bypass the ATM/p53 pathway.

Description: Key Points USP7 is overexpressed and regulates HRR in CLL cells. USP7 inhibition is selectively cytotoxic to CLL cells independently of ATM and p53 and synergizes with chemotherapy.

Description: Current prognostic indicators such as age, sex, and white blood cell count (WBC) fail to identify all children with more aggressive forms of B-precursor acute lymphoblastic leukemia (ALL), and a proportion of patients without poor prognostic indicators still relapse. Results obtained from an analysis of 65 pediatic B-precursor ALL patients indicated that subclone formation leading to clonal diversity, as detected by Ig and T-cell receptor (TCR) gene rearrangements, may represent a very useful prognostic indicator, independent of age, sex, and WBC. Disease-free survival was significantly shorter in those patients showing clonal diversity at presentation. Furthermore, clonal diversity was detected not only in the majority of high-risk patients who relapsed but was also associated with a high probability of relapse in standard-risk patients. Sixty-five percent (13/20) of standard-risk patients who also showed clonal diversity subsequently relapsed, whereas the percentage of relapses among standard-risk patients without clonal diversity was much lower at 19% (7/36). Continued clonal evolution during disease progression is an important feature of aggressive B-precursor ALL. All 5 patients with clonal diversity who were followed up in our study showed a change in the pattern of clonality between presentation and relapse. This implies an important role for clonal diversity as a mechanism of disease progression through the process of clonal variation and clonal selection. © 1998 by The American Society of Hematology.