Acute Lymphoblastic Leukemia Clones Research Articles

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia, however little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CBP) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300, leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using ChIP assay, we demonstrate occupancy by CBP of the survivin promoter, which is decreased by ICG-001 in primary ALL. CBP-mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Abstract 4293: Notch expression expands the pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting self-renewal

Abstract Over 60% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, and the cMYC pathway is also commonly activated in this disease. Notch and cMyc have been shown to collaborate in mouse transgenic models of T-ALL, suggesting that these oncogenes function in parallel pathways to enhance tumor growth; however, the exact role of each in T-ALL is unknown. Here, we show that co-expression of Notch and cMyc in zebrafish T cells significantly enhances T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with cMyc does not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to T-ALLs expressing cMyc alone. Clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch increases the number of T-ALL clones contained within the primary tumor when compared to cMyc expressing leukemais. A large portion of T-ALL clones present in primary Notch and Notch/cMyc leukemias cannot transfer disease into transplanted animals, while all cMyc-alone expressing T-ALLs clones are capable of engraftment and reinitiation of leukemia. Large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that primary T-ALLs expressing either Notch or Notch/cMyc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, both the Notch and Notch/cMyc leukemias exhibit similar leukemia-initiating frequency as cMyc-induced T-ALLs, indicating that Notch signaling does not enhance self-renewal of T-ALL initiating cells. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, cMyc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells, accounting for the longer latency that likely reflects the acquisition of additional genetic changes in clones. Our data may explain why a subset of relapsed human T-ALLs develop from an underrepresented clone found in the primary leukemia, in that primary human T-ALLs likely have a large pool of pre-malignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2011-4293

The Repertoire of Gene Rearrangements in Acute Lymphoblastic Leukemia.

Rearrangements of the immunoglobulin and T-cell receptor genes provide molecular markers for clones in acute lymphoblastic leukemia (ALL). Determination of the repertoire of gene rearrangements in ALL aids in understanding the clonal biology of the disease and provides molecular markers which can be used to quantify minimal residual disease (MRD).We have developed a sensitive PCR-based method for analysing the repertoire of immunoglobulin heavy chain (IgH) rearrangements in ALL. Multiple parallel quantitative PCR's are performed in microplates using different segment-specific primers in different wells in order to determine the individual V (D) and J segments utilised by each rearrangement. The number of rearrangements detected in 18 children and 10 adults with ALL is shown in the table:VDJ rearrangementsDJ rearrangementsNo. of rearrangements123412Childhood ALL0113211Adult ALL7201Since each PCR well contained only 2 ng of DNA, more sensitive repertoire analysis was also performed in samples from 10 of the children and 4 of the adults by using 100 ng of DNA in an initial preamplification, which involved a multiplexed PCR containing primers for all leader and J sequences of the IgH gene and which thus amplified all immunoglobulin sequences. The IgH repertoire of the amplified material was then analysed. This two-step approach should theoretically enable detection of clones which comprise down to approximately 10−4 the leukemic population. It detected all rearrangements previously detected by one-step repertoire analysis and, in addition, it detected 0–5 (mean 1.2) rearrangements marking small clones in childhood ALL and 0–3 (mean 1.0) rearrangements marking small clones in adult ALL. Sequencing showed that most, but not all, small clones had a lineage relationship to the dominant clone present in the leukemic population.Repertoire analysis of IgH rearrangements is a promising technique for identifying molecular markers for measurement of MRD in B-ALL, particularly childhood ALL, since:–it is conceptually simple and relatively quick–it detects IgH rearrangements with high efficiency, probably higher than that of current techniques.–IgH rearrangements are the best markers to use for measurement of MRD owing to the specificity and sensitivity that they provide–an enhanced ability to identify markers for both large and small leukemic clones may improve the identification of patients prone to relapse.