Access Type

Open Access Dissertation

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Larry H. Matherly

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric ALL cases and is associated with early relapse and inferior outcome. The poorer prognosis of T-ALL compared to B-precursor ALL may in part reflect the lack of unique features on which to base therapy. NOTCH1 mutations are of particular interest since these were reported in 37-71% of T-ALLs. The prognostic value of NOTCH1 mutations remains controversial as both favorable and unfavorable associations were reported, whereas in other studies, there were no associations between NOTCH1 mutations and treatment outcome. We explored the impact of mutations in NOTCH1, FBW7 and PTEN on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia. We identified a high frequency of mutations in NOTCH1 (16 patients), FBW7 (5 patients) and PTEN (26 patients) in a well defined cohort of 47 pediatric T-ALL specimens. NOTCH1 mutations showed a 1.3-3.3-fold increase in activation over wild-type NOTCH1 in reporter assays; mutant FBW7 resulted in further augmentation of NOTCH1 activity. NOTCH1 and FBW7 mutations were accompanied by increases in median transcripts for NOTCH1 target genes (HES1, DELTEX1 and cMYC). However, none of these mutations were associated with treatment outcome. Increased HES1, DELTEX1 and cMYC transcript levels were associated with significant increases in the transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltransferase, Bcl-2 and dihydrofolate reductase. Our results suggest (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL and (2) that, depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.

Relapse is the most common caused of off-therapy events and is responsible for the majority of ALL treatment failures. Relapse can arise from the (i) the induction of resistance via acquisition of new genetic alterations after diagnosis, (ii) the selection and expansion of an already present resistant-subpopulation at the time of diagnosis, or very rarely as (iii) a secondary, de novo ALL. To determine the contribution of genetic alteration to the development of relapse in T-ALL, we assessed the frequency of mutations in NOTCH1 alone or in combination with mutations in FBW7 and PTEN at the time of diagnosis and relapse in 11 paired clinical T-ALL specimens. We observed that the 7 patients harboring mutations in NOTCH1 and/or PTEN at some stage in their disease had a longer remission period (13 months vs. 5.5 months), and were typically diagnosed at an early age (120 months vs. 132 months). In these 7 patients, nearly 70% of relapse appeared to be associated with the emergence of a new leukemic clone, an assumption made by the presence of a new mutation or loss of a mutation at relapse. Using real-time PCR techniques with specific hybridization probes, we were able to determine that the leukemic clone for one patient was present at the time of diagnosis, but at a very low expression level. This suggests that the clone responsible for relapse was resistant to the initial chemotherapy treatment. For another patient, the relapse clone could not be detected at diagnosis, suggesting that it was induced following chemotherapy. This study strongly warrants future studies with a larger patient cohort to systematically identify specific hallmarks of relapse.

NOTCH1 is a potentially attractive therapeutic target for T-ALL since constitutively activating effects of mutant NOTCH1 can be abolished with -secretase inhibitors (GSIs). Because of possible effects of GSIs on other cellular targets in addition to NOTCH1, we explored shRNA knockdown of NOTCH1 to identify novel NOTCH1-regulated genes that may serve as prognostic indicators or therapeutic targets in T-ALL. NOTCH1 expression was knockeddown in Jurkat T-ALL cells using lentivirus expressing shRNAs for NOTCH1 or a non-targeted control (J.ntc) sequence. NOTCH1 knockdown was verified using western blots to measure activated NOTCH1 (ICN1) protein levels, and real-time RT-PCR to measure transcript levels of known NOTCH1 targets (e.g., HES1). Two clonal sublines (J.N1KD 2-4 and J.N1KD 2-7) were identified with significantly decreased expression of NOTCH1 compared to J.ntc. The J.N1KD 2-4 and J.N1KD 2-7 sublines showed minimal changes in cell growth, cell cycle progression and apoptosis. To characterize genotypic changes accompanying NOTCH1 knockdown, we performed microarray analysis with Agilent Whole Genome oligonucleotide microarrays and microRNA (miR) HumanV2 arrays. The microarray identified Rictor, a key component to in the mTOR2 complex, as a novel downstream target of NOTCH1 signaling. Upon NOTCH1 inhibition, an increase in the expression of Rictor was observed, both at the transcript and protein levels. Initial computational analysis of the Rictor promoter suggests that NOTCH1 may regulate its expression directly (via RBPJ) or indirectly (via HES1). The miR array identified 20 miRs in J.N1KD 2-4 and J.N1KD 2-7 cells with altered expression compared to J.ntc greater than 1.5-fold (p〈0.05) and ranging from 3-to10-fold. miRs hsa-Let-7e, hsa-miR-125a-5p and hsa-miR-99b, reportedly derived from a polycistronic transcript, were decreased 10-fold accompanying NOTCH1 knockdown. Using miR qPCR, we confirmed decreased levels of hsa-miR-125a-5p and hsa-miR-99b in the J.N1KD 2-4 and J.N1KD 2-7 sublines. In conclusion, we have developed novel T-ALL cell line models to study the impact of decreased NOTCH1 levels and activity independent of GSI treatment. Our results implicate NOTCH1 in regulating levels of Rictor and hsa-miR-125a-5p, and suggest that caution may be warranted in targeting NOTCH1 with GSIs in the therapy of T-ALL, reflecting the potential promotion of cell survival via the upregulation of Rictor. The downstream effect of regulating hsa-miR-125a-5p has yet to be determined.

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