Aneela Majid

Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-X(L). ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC(50) approximately 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737-induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-X(L) and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737-related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.

Mutation Status of the Residual ATM Allele Is an Important Determinant of the Cellular Response to Chemotherapy and Survival in Patients With Chronic Lymphocytic Leukemia Containing an 11q Deletion

PURPOSE The ataxia telangiectasia mutated (ATM) gene is located on chromosome 11q and loss of this region is common in B-cell chronic lymphocytic leukemia (CLL). Our aim was to determine if CLL tumors with a chromosome 11q deletion might be divided into two subgroups based on the status of the remaining ATM allele. METHODS The sequence of the residual ATM allele was determined in 72 CLLs with an 11q deletion. This was related to the cellular response to irradiation or cytotoxic drug exposure in vitro and clinical outcome. RESULTS We show that the residual ATM allele is mutated in 36% of CLLs with an 11q deletion and that these leukemias demonstrate an impaired cellular response to irradiation or cytotoxic drug exposure in vitro. Inactivation of the second ATM allele was associated with a reduction in patient survival beyond that already dictated by the presence of an 11q deletion (P = .0283). Furthermore, we demonstrate that ATM mutations may arise during the evolution of an 11q deleted subclone and are associated with its expansion. CONCLUSION CLL with 11q deletion can be divided into two subgroups based on the integrity of the residual ATM allele. Patients with complete loss of ATM function, due to biallelic ATM defects, have defective responses to cytotoxic chemotherapeutics in vitro and a poorer clinical outcome. ATM mutant subclones can develop during an individuals disease course and give rise to additional expansion of the 11q deleted subclone.

Chronic lymphocytic leukemic cells exhibit apoptotic signaling via TRAIL-R1

Clinical trials have been initiated with Apo2L/TRAIL (Genentech) and agonistic mAbs to TRAIL receptors, -R1 and -R2 (Human Genome Sciences). The apoptosis-inducing ability of these mAbs and different TRAIL preparations, in the presence or absence of histone deacetylase inhibitors (HDACi), varied markedly against primary chronic lymphocytic leukaemia (CLL) cells and various tumor cell lines, demonstrating an unanticipated preferential apoptotic signaling via either TRAIL-R1 or -R2. Contrary to literature reports that TRAIL-induced apoptosis occurs primarily via signaling through TRAIL-R2, CLL cells, in the presence of HDACi, undergo predominantly TRAIL-R1-mediated apoptosis. Consequently, Apo2L/TRAIL, which signals primarily through TRAIL-R2, is virtually devoid of activity against CLL cells. To maximize therapeutic benefit, it is essential to ascertain whether a primary tumor signals via TRAIL-R1/-R2, prior to initiating therapy. Thus combination of an agonistic TRAIL-R1 Ab and an HDACi, such as the anticonvulsant sodium valproate, could be of value in treating CLL.

A genome-wide association study identifies multiple susceptibility loci for chronic lymphocytic leukemia.

Genome-wide association studies (GWAS) of chronic lymphocytic leukemia (CLL) have shown that common genetic variation contributes to the heritable risk of CLL. To identify additional CLL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1,739 individuals with CLL (cases) and 5,199 controls with validation in an additional 1,144 cases and 3,151 controls. A combined analysis identified new susceptibility loci mapping to 3q26.2 (rs10936599, P = 1.74 × 10−9), 4q26 (rs6858698, P = 3.07 × 10−9), 6q25.2 (IPCEF1, rs2236256, P = 1.50 × 10−10) and 7q31.33 (POT1, rs17246404, P = 3.40 × 10−8). Additionally, we identified a promising association at 5p15.33 (CLPTM1L, rs31490, P = 1.72 × 10−7) and validated recently reported putative associations at 5p15.33 (TERT, rs10069690, P = 1.12 × 10−10) and 8q22.3 (rs2511714, P = 2.90 × 10−9). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to CLL.

A subset of Binet stage A CLL patients with TP53 abnormalities and mutated IGHV genes have stable disease

TP53 abnormalities consistently emerge as the most significant adverse prognostic factor in multivariate analyses of both prospective and retrospective studies in early and advanced chronic lymphocytic leukaemia (CLL).1 In view of the higher response rates often achieved with alemtuzumab-containing regimens compared with alkylating agent and/or purine analogue therapy2 and the possibility of long-term disease-free survival following allogeneic transplantation in some patients, there is an increasing consensus for screening for TP53 abnormalities in CLL patients who fulfil the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) recommendations for initiating therapy.

TRAIL signals to apoptosis in chronic lymphocytic leukaemia cells primarily through TRAIL-R1 whereas cross-linked agonistic TRAIL-R2 antibodies facilitate signalling via TRAIL-R2

Tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL), a member of the TNF family, which is being developed as an anti‐tumour agent due to its selective toxicity to tumour cells, induces apoptosis by binding to two membrane‐bound receptors, TRAIL‐R1 and TRAIL‐R2. Clinical trials have been initiated with various preparations of TRAIL as well as agonistic monoclonal antibodies to TRAIL‐R1 and TRAIL‐R2. Previously we reported that prior treatment of primary chronic lymphocytic leukaemia (CLL) cells with histone deacetylase inhibitors was required to sensitize CLL cells to TRAIL and, using various receptor‐selective TRAIL mutant ligands, we demonstrated that CLL cells signalled to apoptosis primarily through TRAIL‐R1. Some, but not all, agonistic TRAIL‐receptor antibodies require cross‐linking in order to induce apoptosis. The present study demonstrated that CLL cells can signal to apoptosis through the TRAIL‐R2 receptor, but only after cross‐linking of the agonistic TRAIL‐R2 antibodies, LBY135 and lexatumumab (HGS‐ETR2). In contrast, signalling through TRAIL‐R1 by receptor‐selective ligands or certain agonistic antibodies, such as mapatumumab (HGS‐ETR1), occurs in the absence of cross‐linking. These results further highlight important differences in apoptotic signalling triggered through TRAIL‐R1 and TRAIL‐R2 in primary tumour cells. Such information is clearly important for the rational optimisation of TRAIL therapy in primary lymphoid malignancies, such as CLL.

Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells

Background Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells. Design and Methods Using western blot analysis, we monitored the regulation of BCL2 family members, proteins of the unfolded protein response (endoplasmic reticulum stress response) and activation of caspases in relation to induction of apoptosis (measured by annexin-propidium iodide staining and loss of mitochondrial membrane potential) by bortezomib in chronic lymphocytic leukemia cells. Results Bortezomib induced apoptosis through activation of the mitochondrial pathway independently of changes associated with endoplasmic reticulum stress. Perturbation of mitochondria was regulated by a rapid and transcription-independent increase of NOXA protein, which preceded release of cytochrome c, HtrA2, Smac and activation of caspase-9 and −3. NOXA had a short half life (~ 1–2 h) and was ubiquitinated on at least three primary lysine residues, resulting in proteasomal-dependent degradation. Down-regulation of NOXA, using short interfering RNA in chronic lymphocytic leukemia cells, decreased bortezomib-induced apoptosis. Finally bortezomib when combined with seliciclib resulted in a stronger and earlier increase in NOXA protein, caspase-3 cleavage and induction of apoptosis in chronic lymphocytic leukemia cells. Conclusions These results highlight a critical role for NOXA in bortezomib–induced apoptosis in chronic lymphocytic leukemia cells and suggest that this drug may become more efficient for the treatment of chronic lymphocytic leukemia if combined with other agents able to interfere with the basal levels of MCL1.

A novel functional assay using etoposide plus nutlin-3a detects and distinguishes between ATM and TP53 mutations in CLL

A novel functional assay using etoposide plus nutlin-3a detects and distinguishes between ATM and TP53 mutations in CLL

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.