Naina Patel

TEL Deletion Analysis Supports a Novel View of Relapse in Childhood Acute Lymphoblastic Leukemia

Purpose: TEL (ETV6)-AML1 (RUNX1) chimeric gene fusions are frequent genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). They often arise prenatally as early events or initiating events and are complemented by secondary postnatal genetic events of which deletion of the non-rearranged, second TEL allele is the most common. This consistent sequence of molecular pathogenesis facilitates an analysis of the clonal origins of relapse in this leukemia, which has some unusual clinical features. Experimental Design: We compared the boundaries, by microsatellite mapping, of TEL deletions at relapse versus diagnosis in 15 informative patients. Moreover, we compared the relatedness of diagnostic and relapse clones using immunoglobulin and T-cell receptor genes rearrangements and clonotypic TEL-AML1 genomic fusion. Results: Five patients retained the apparent same size TEL deletion, seven had larger deletions, and three had smaller deletions at relapse. In all of the cases evaluated, the clonal relatedness of diagnostic and relapse cells was confirmed by the retention of clonotypic TEL-AML1 genomic sequence and/or at least one identical immunoreceptor gene rearrangement. Conclusions: These data provide further evidence that TEL deletions are secondary to TEL-AML1 fusions in ALL. They are compatible with the novel idea that in at least some cases of childhood ALL, remission occurs with persistence of a preleukemic “fetal” clone, and subsequent relapse reflects the emergence of a new subclone from this reservoir after an independent “second hit,” i.e., independent TEL deletion. To our knowledge, the study is the most extensive and comprehensive analysis of the relationship between diagnostic and relapse clones in childhood ALL presented thus far.

Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity

Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase.

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL.

Prospective gene expression analysis accurately subtypes acute leukaemia in children and establishes a commonality between hyperdiploidy and t(12;21) in acute lymphoblastic leukaemia

We have prospectively analysed and correlated the gene expression profiles of children presenting with acute leukaemia to the Royal London and Great Ormond Street Hospitals with morphological diagnosis, immunophenotype and karyotype. Total RNA extracted from freshly sorted blast cells was obtained from 84 lymphoblastic [acute lymphoblastic leukaemia (ALL)], 20 myeloid [acute myeloid leukaemia (AML)] and three unclassified acute leukaemias and hybridised to the high density Affymetrix U133A oligonucleotide array. Analysis of variance and significance analysis of microarrays was used to identify discriminatory genes. A novel 50‐gene set accurately identified all patients with ALL and AML and predicted for a diagnosis of AML in three patients with unclassified acute leukaemia. A unique gene set was derived for each of eight subtypes of acute leukaemia within our data set. A common profile for children with ALL with an ETV6–RUNX1 fusion, amplification or deletion of ETV6, amplification of RUNX1 or hyperdiploidy with an additional chromosome 21 was identified. This suggests that these rearrangements share a commonality in biological pathways that maintains the leukaemic state. The gene TERF2 was most highly expressed in this group of patients. Our analyses demonstrate that not only is microarray analysis the single most effective tool for the diagnosis of acute leukaemias of childhood but it has the ability to identify unique biological pathways. To further evaluate its prognostic value it needs to be incorporated into the routine diagnostic analysis for large‐scale clinical trials in childhood acute leukaemias.

Differential epigenetic reprogramming in response to specific endocrine therapies promotes cholesterol biosynthesis and cellular invasion

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients.

Expression profile of wild-type ETV6 in childhood acute leukaemia

Summary. Comparative expression analysis of wild‐typeETV6 in the disease state showed an absence of expression in ETV6–CBFA2 acute lymphoblastic leukaemia (ALL) when compared with non‐ETV6–CBFA2 ALL and acute myeloid leukaemia. Fluorescent in‐situ hybridization and loss of heterozygosity studies showed that 73% of the ETV6–CBFA2 samples had a fully or partially deleted second ETV6 allele, explaining the lack of wild‐type expression in these patients. Although the second ETV6 allele was identified in the remaining patients, no ETV6 expression was detected. These observations support the hypothesis that loss of ETV6 expression is a critical secondary event for leukaemogenesis in ETV6–CBFA2 ALL.

Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia

The t(10;11)(p13;q14–21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927–2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

Loss of O6-methylguanine-DNA methyltransferase confers collateral sensitivity to carmustine in topoisomerase II-mediated doxorubicin resistant triple negative breast cancer cells

Triple-negative breast cancer is characterized by aggressive tumours whose cells lack oestrogen and progesterone receptors and do not over-express HER2. It accounts for approximately 10-15% of breast cancer cases. We sought to generate a cellular model of chemotherapy drug resistance for this type of disease to provide the tools for the development of new therapies. Doxorubicin is a component of some chemotherapy regimes used to treat this form of cancer but resistance preventing disease eradication frequently occurs, mainly due to over-expression of drug transporters such as P-glycoprotein. CALDOX cells were generated by exposure of CAL51 to doxorubicin. Resistance to doxorubicin did not involve drug transporters, as the both parental and resistant cells accumulated doxorubicin to comparable levels. CALDOX cells had slower proliferation rate and an extended G1 cell cycle stage than the parental line, mainly due to an intrinsic activation of CDNK1 (p21), but this cell cycle block was not involved in the mechanism of resistance. CALDOX cells had reduced levels of TOP2A (topoisomerase IIα) and were cross resistant to the topoisomerase II inhibitors etoposide and mitoxantrone. CALDOX cells showed collateral sensitivity to carmustine due to the lack of O⁶-methylguanine-DNA-methyltransferase (MGMT) expression, related to the hypermethylation of its promoter. The collateral sensitivity of CALDOX cells to carmustine provides the rationale to evaluate MGMT promoter methylation status to design better therapeutic strategies for triple negative breast cancer.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-α and the transcriptional repressor PLZF

Estrogen receptor α (ERα) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERα plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERα and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERα is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERα expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERα is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

Cryptic rearrangement involving MLL and AF10 occurring in utero

Chimaeric fusion genes created by nonrandom chromosomal translocations are characteristic of childhood acute lymphoblastic (ALL) and myeloid (AML) leukaemias. Although the mechanisms leading to these chromosomal rearrangements are unclear, careful analysis of the primary genomic fusion sequence has suggested a number of possible mechanisms. These include, aberrant VDJ recombinase activity, topoisomerase II-mediated breakage followed by erroneous end joining, illegitimate recombination mediated by repetitive DNA sequences, proteins that bind to specific DNAbinding sites and errors in nonhomologous end joining (NHEJ) following double-stranded DNA breaks. Using polymerase chain reaction (PCR) -based techniques on DNA extracted from neonatal Guthrie cards, belonging to children diagnosed with leukaemia, it has been possible to show that some of these fusions occur prenatally. Fusions detected to date include the MLL-AF4 fusion in infantile ALL; the TEL-AML1 in childhood ALL and the AML1-ETO and MLL-CALM in childhood AML. Analysis of cells obtained from normal cord blood have shown that TEL-AML1 and AML1-ETO fusions are detected in the normal population at birth at a frequency that is a 100 times higher than the incidence of corresponding leukaemia. Thus, while the events leading to the formation of chimaeric fusion genes occur in utero, only a few develop clinically overt leukaemia at a later date in early childhood. This suggests that additional postnatal genetic hits are required in these preleukaemic cells. Murine models of TEL-AML1 and AML1-ETO support this hypothesis, as the transgenes are insufficient themselves to induce leukaemia. We extend these observations by identifying a child with AML carrying a prenatally acquired MLL-AF10 fusion. A 24-month-old boy presented with an acute history of fever and parotid enlargement. He was found to have subcutaneous nodules, generalised lymphadenopathy, splenomegaly and pancytopenia. Histopathological examination of bone marrow (BM) aspirate, trephine, lymph node (LN) and skin nodule did not reveal any features of a malignancy. Symptoms and signs regressed spontaneously and the child was kept under observation. After 6 weeks, all symptoms and signs recurred. This time, the marrow was infiltrated with 70% FAB M5 myeloid blasts and the LN biopsy showed a heavy infiltrate of malignant myeloid cells. He was started on the Medical Research Council AML 12 Trial and went into remission after the first course of chemotherapy. After 18 months, he is alive and well. Cytogenetic analyses of the lymph node sample obtained at initial presentation detected the presence of an abnormal clone 46,XY, dic(1;19)(q10;q13) in a background of morphologically normal cells. This clone was also seen in 1/100 cells obtained from the BM sample obtained at the same time. Presence of this abnormal clone was confirmed from both BM and LN samples when the child represented with florid disease. At this time, routine screening of the same metaphase spread using fluorescent in situ hybridisation (FISH) with a probe for the MLL gene (Vysis, Richmond, UK) revealed an insertion of MLL into 10p12 (Figure 1a). No structural chromosomal alterations involving 11q23 were detected by conventional Gbanding and this insertion could not be detected by the use of whole-chromosome 11 paint (Figure 1b). The MLL and AF10 genes are oriented in the opposite direction on their respective chromosomes. Thus, complex chromosomal rearrangements, requiring at least three breaks are required for an in-frame 50MLL-30AF10 fusion to occur. Although these rearrangements are usually visible at the cytogenetic level, as is illustrated by this case, they can occasionally be cryptic underlining the importance of using FISH in addition to G-banding to characterise leukaemia-associated cytogenetic changes. We have previously described the t(10;11)(p12;q23) to result in an MLL-AF10 fusion. RNA was extracted from diagnostic BM cells, and a nested reverse transcriptase polymerase chain reaction (RTPCR) was performed to detect the MLL-AF10 fusion transcript as previously described. First round PCR was carried out with MLL ex 5F and AF1