Jules P.P. Meijerink

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

γ-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

γ-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia

Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2–like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Krüppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.Summary Gamma-secretase inhibitors (GSIs) block the activation of oncogenic NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor auto-up-regulation and induced apoptotic cell death through induction of BIM expression. GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of Klf4, a negative regulator of cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of Ccnd2 and protected mice from developing intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL.

A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.

Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia.

Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R–mediated signaling in T-ALL.

Integrated Transcript and Genome Analyses Reveal NKX2-1 and MEF2C as Potential Oncogenes in T Cell Acute Lymphoblastic Leukemia

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.

Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia

We identified a duplication of the MYB oncogene in 8.4% of individuals with T cell acute lymphoblastic leukemia (T-ALL) and in five T-ALL cell lines. The duplication is associated with a threefold increase in MYB expression, and knockdown of MYB expression initiates T cell differentiation. Our results identify duplication of MYB as an oncogenic event and suggest that MYB could be a therapeutic target in human T-ALL.

PHF6 mutations in T-cell acute lymphoblastic leukemia

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is importantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease.

The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is mostly characterized by specific chromosomal abnormalities, some occurring in a mutually exclusive manner that possibly delineate specific T-ALL subgroups. One subgroup, including MLL-rearranged, CALM-AF10 or inv (7)(p15q34) patients, is characterized by elevated expression of HOXA genes. Using a gene expression-based clustering analysis of 67 T-ALL cases with recurrent molecular genetic abnormalities and 25 samples lacking apparent aberrations, we identified 5 new patients with elevated HOXA levels. Using microarray-based comparative genomic hybridization (array-CGH), a cryptic and recurrent deletion, del (9)(q34.11q34.13), was exclusively identified in 3 of these 5 patients. This deletion results in a conserved SET-NUP214 fusion product, which was also identified in the T-ALL cell line LOUCY. SET-NUP214 binds in the promoter regions of specific HOXA genes, where it interacts with CRM1 and DOT1L, which may transcriptionally activate specific members of the HOXA cluster. Targeted inhibition of SET-NUP214 by siRNA abolished expression of HOXA genes, inhibited proliferation, and induced differentiation in LOUCY but not in other T-ALL lines. We conclude that SET-NUP214 may contribute to the pathogenesis of T-ALL by enforcing T-cell differentiation arrest.

Direct Reversal of Glucocorticoid Resistance by AKT Inhibition in Acute Lymphoblastic Leukemia

Glucocorticoid resistance is a major driver of therapeutic failure in T cell acute lymphoblastic leukemia (T-ALL). Here, we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid-induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy, and effectively reverses glucocorticoid resistance in vitro and in vivo.

Deletion of the protein tyrosine phosphatase gene PTPN2 in T-cell acute lymphoblastic leukemia.

PTPN2 (protein tyrosine phosphatase non-receptor type 2, also known as TC-PTP) is a cytosolic tyrosine phosphatase that functions as a negative regulator of a variety of tyrosine kinases and other signaling proteins. In agreement with its role in the regulation of the immune system, PTPN2 was identified as a susceptibility locus for autoimmune diseases. In this work, we describe the identification of focal deletions of PTPN2 in human T-cell acute lymphoblastic leukemia (T-ALL). Deletion of PTPN2 was specifically found in T-ALLs with aberrant expression of the TLX1 transcription factor oncogene, including four cases also expressing the NUP214-ABL1 tyrosine kinase. Knockdown of PTPN2 increased the proliferation and cytokine sensitivity of T-ALL cells. In addition, PTPN2 was identified as a negative regulator of NUP214-ABL1 kinase activity. Our study provides genetic and functional evidence for a tumor suppressor role of PTPN2 and suggests that expression of PTPN2 may modulate response to treatment.