Search

Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34+ stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients, we now demonstrate that some, but not all, of these parameters correlate with subsequent clinical response to IM therapy. CD34+ cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P < .001) and direct sequencing of cloned transcripts from CD34+ cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders, P < .003). In contrast, CD34+ cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly, one BCR-ABL mutation (V304D), predicted to destabilize the interaction between p210BCR-ABL and IM, was detectable in 14 of 20 patients. T315I mutant CD34+ cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus, 2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management.

(Abelson helper integration site-1) is a novel oncogene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma as a candidate cooperate oncogene. The Ahi-1 protein has a SH3 domain, multiple SH3 binding sites and WD-repeat domains, suggesting novel signaling activities. We have recently demonstrated that AHI-1 is highly deregulated in human leukemic cells, particularly in BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML). Overexpression of Ahi-1 in primitive hematopoietic cells confers a growth advantage in vitro and induces leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that plays a major role in the genesis of CML. Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL-transduced lin−CD34+ human cord blood cells and leukemic stem/progenitor cells from CML patients reduces their growth autonomy in vitro. Interestingly, a direct physical interaction between AHI-1 and BCR-ABL at endogenous levels has been identified in CML cells and this interaction complex further mediates tyrosine kinase inhibitor response/resistance of CML stem/progenitor cells. To further investigate regulatory roles of Ahi-1 in mediating BCR-ABL transforming activities and altered signaling, we have now evaluated co-operative effects of Ahi-1 in a BCR-ABL inducible BaF3 cell line model in which the level of expression of p210BCR-ABL can be variably down-regulated by exposure to doxycycline (Dox). These experiments showed that reduction in BCR-ABL protein expression in the presence of Dox resulted in a corresponding decrease in growth factor independence both in liquid suspension cultures and in semi-solid cultures and an increase in Annexin V+ apoptotic cells in vitro. Interestingly, stable co-expression of Ahi-1 in BCR-ABL inducible BaF3 cells under these stringent conditions enabled them to grow continuously in liquid suspension culture, with fewer Annexin V+ apoptotic cells, and to produce more factor independent CFCs than cells transduced with BCR-ABL alone (10–30 fold). Strikingly, Ahi-1 co-transduced cells also displayed greater resistance to imatinib and, in the presence of IL-3, produced as many CFCs as were produced by the same cells without treatment of imatinib. In contrast, BCR-ABL-transduced cells alone showed a significant reduction of CFC output in response to imatinib. Western blot analysis further demonstrated that co-expression of Ahi-1 in BCR-ABL inducible cells resulted in sustained phosphorylation of BCR-ABL and enhanced activation of JAK2/STAT5 compared to BCR-ABL inducible cells alone when BCR-ABL expression was downregulated in the presence of Dox. Moreover, physical interaction between Ahi-1 and BCR-ABL was demonstrated by co-IP studies in Ahi-1 co-expressed BCR-ABL inducible cells. Taken together, these results provide direct evidence of the regulatory role of Ahi-1 in BCR-ABL-mediated transformation and imatinib response that is associated with altered BCR-ABL phosphorylation and JAK2/STAT5 activation.

Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P