Jennifer Hüllein

Evolution of DNA methylation is linked to genetic aberrations in chronic lymphocytic leukemia

Although clonal selection by genetic driver aberrations in cancer is well documented, the ability of epigenetic alterations to promote tumor evolution is undefined. We used 450k arrays and next-generation sequencing to evaluate intratumor heterogeneity and evolution of DNA methylation and genetic aberrations in chronic lymphocytic leukemia (CLL). CLL cases exhibit vast interpatient differences in intratumor methylation heterogeneity, with genetically clonal cases maintaining low methylation heterogeneity and up to 10% of total CpGs in a monoallelically methylated state. Increasing methylation heterogeneity correlates with advanced genetic subclonal complexity. Selection of novel DNA methylation patterns is observed only in cases that undergo genetic evolution, and independent genetic evolution is uncommon and is restricted to low-risk alterations. These results reveal that although evolution of DNA methylation occurs in high-risk, clinically progressive cases, positive selection of novel methylation patterns entails coevolution of genetic alteration(s) in CLL.

p53-dependent non-coding RNA networks in chronic lymphocytic leukemia.

Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Whereas p53 targets are used to identify patient subgroups with impaired p53 function, a comprehensive assessment of non-coding RNA targets of p53 in CLL is missing. We exploited the impaired transcriptional activity of mutant p53 to map out p53 targets in CLL by small RNA sequencing. We describe the landscape of p53-dependent microRNA/non-coding RNA induced in response to DNA damage in CLL. Besides the key p53 target miR-34a, we identify a set of p53-dependent microRNAs (miRNAs; miR-182-5p, miR-7-5p and miR-320c/d). In addition to miRNAs, the long non-coding RNAs (lncRNAs) nuclear enriched abundant transcript 1 (NEAT1) and long intergenic non-coding RNA p21 (lincRNA-p21) are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Induction of NEAT1 and lincRNA-p21 are closely correlated to the induction of cell death after DNA damage. We used isogenic lymphoma cell line models to prove p53 dependence of NEAT1 and lincRNA-p21. The current work describes the p53-dependent miRNome and identifies lncRNAs NEAT1 and lincRNA-p21 as novel elements of the p53-dependent DNA damage response machinery in CLL and lymphoma.

Targeted resequencing for analysis of clonal composition of recurrent gene mutations in chronic lymphocytic leukaemia

Recurrent gene mutations contribute to the pathogenesis of chronic lymphocytic leukaemia (CLL). We developed a next‐generation sequencing (NGS) platform to determine the genetic profile, intratumoural heterogeneity, and clonal structure of two independent CLL cohorts. TP53, SF3B1, and NOTCH1 were most frequently mutated (16·3%, 16·9%, 10·7%). We found evidence for subclonal mutations in 67·5% of CLL cases with mutations of cancer consensus genes. We observed selection of subclones and found initial evidence for convergent mutations in CLL. Our data suggest that assessment of (sub)clonal structure may need to be integrated into analysis of the mutational profile in CLL.

Recurrent CDKN1B (p27) mutations in hairy cell leukemia.

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B (p27). Targeted deep sequencing of CDKN1B in a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1B mutations.

The impact of SF3B1 mutations in CLL on the DNA-damage response

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

BRAF inhibition in hairy cell leukemia with low-dose vemurafenib.

The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care.

Leflunomide Induces Apoptosis in Fludarabine-Resistant and Clinically Refractory CLL Cells

Purpose: Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells. Experimental Design: CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726. Results: Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4–induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4–activated (“resistant”) CLL cells was achieved with higher concentrations (IC50: 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC50: 56 μg/mL). Conclusion: We thus show that A771726 overcomes CD40L/IL-4–mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells. Clin Cancer Res; 18(2); 417–31. ©2011 AACR.

Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

Significance B-cell receptor (BCR) signaling promotes the survival of malignant B cells, such as Burkitt’s lymphoma (BL) and the activated B-cell–like subtype of diffuse large B-cell lymphoma (ABC-DLBCL). In contrast to ABC-DLBCL, which depends on chronic activation of the BCR, BL cells rely on tonic BCR signaling that is antigen-independent. Elucidation and systematic comparison of tonic and activated BCR signaling led to the identification of novel signaling effectors, including ACTN4 and ARFGEF2, which were identified as regulators of BL-cell survival. Beyond its relevance to the understanding of BL pathogenesis and the development of targeted therapies, our study complements the general understanding of BCR-induced processes also in physiological settings. Burkitts lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments.

Next-generation sequencing of cancer consensus genes in lymphoma

Abstract Sensitive identification of mutations in genes related to the pathogenesis of cancer is a prerequisite for risk-stratified therapies. Next-generation sequencing (NGS) in lymphoma has revealed genetic heterogeneity which makes clinical translation challenging. We established a 454-based targeted resequencing platform for robust high-throughput sequencing from limited material of patients with lymphoma. Hotspot mutations in the most frequently mutated cancer consensus genes were amplified in a two-step multiplex-polymerase chain reation (PCR) which was optimized for homogeneous coverage of all regions of interest. We show that targeted resequencing based on NGS technologies allows highly sensitive detection of mutations and assessment of clone size. The application of this or similar techniques will help the development of genotype-specific treatment approaches in lymphoma.

Genomic analysis of hairy cell leukemia identifies novel recurrent genetic alterations

Classical hairy cell leukemia (cHCL) is characterized by a near 100% frequency of the BRAFV600E mutation, whereas ∼30% of variant HCLs (vHCLs) have MAP2K1 mutations. However, recurrent genetic alterations cooperating with BRAFV600E or MAP2K1 mutations in HCL, as well as those in MAP2K1 wild-type vHCL, are not well defined. We therefore performed deep targeted mutational and copy number analysis of cHCL (n = 53) and vHCL (n = 8). The most common genetic alteration in cHCL apart from BRAFV600E was heterozygous loss of chromosome 7q, the minimally deleted region of which targeted wild-type BRAF, subdividing cHCL into those hemizygous versus heterozygous for the BRAFV600E mutation. In addition to CDKN1B mutations in cHCL, recurrent inactivating mutations in KMT2C (MLL3) were identified in 15% and 25% of cHCLs and vHCLs, respectively. Moreover, 13% of vHCLs harbored predicted activating mutations in CCND3 A change-of-function mutation in the splicing factor U2AF1 was also present in 13% of vHCLs. Genomic analysis of de novo vemurafenib-resistant cHCL identified a novel gain-of-function mutation in IRS1 and losses of NF1 and NF2, each of which contributed to resistance. These data provide further insight into the genetic bases of cHCL and vHCL and mechanisms of RAF inhibitor resistance encountered clinically.