Hedgehog Signaling Is Useful as a Novel Molecular Marker for Predicting Relapse and Resistance During Chronic Myeloid Leukemia Treatment.

Abstract 1215

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a Leukemic Stem Cell (LSC) clone carrying a Philadelphia translocation, which outgrows the non-malignant haematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, are the gold standard for CML treatment since each one shows an impressive rates of complete cytogenetic and molecular response in chronic phase (CP) CML. However the major problem concerning the final efficacy of TKIs therapy is that the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML LSC not effectively targeted by TKIs. Therefore the molecular monitoring not always provide a sufficiently precise evaluation of patients to allow the appropriate choice of clinical interventions. Accordingly, it is necessary to monitor the appearance and increase of LSCs to identify and to treat quickly the fundamental responsible for relapse. Thus, we focused Hedgehog (Hh) signalling which has been proved essential for maintenance of cancer stem cells in myeloid leukaemia. Notably recent studies reported that the expansion of BCR-ABL positive leukemic stem cell is dependent on Hh pathway activation. Here, we analyzed the mRNA levels of Smoothened (SMO), a seven-transmembrane domain receptor protein, and Ptch1, a surface receptor regulator of SMO, in 20 CP-CML patients (8 High, 4 Intermediate and 8 Low Sokal Risk respectively) at diagnosis and during the follow-up. Using RT-PCR, in diagnosis setting, we proved that 60% of patients (bone marrow samples) showed Hh signalling significantly activated compared to CD34+ cells from healthy donors. In detail 75% (6/8) of High Sokal Risk CML patients showed an up-regulation of Smo and a down-regulation of Ptch1 mRNA levels, suggestive of active Hh signalling at diagnosis. Conversely Low and Intermediate Sokal Risk CML patients did not show features of Hh activation. Finally we monitored, during the follow-up, the mRNA levels of Smo and Ptch1 together with BCR-ABL to assess the kind of relationship between these parameters. Interestingly we noticed a direct correlation between the increase of BCR-ABL mRNA levels and signs of Hh activity (Smo mRNA increase level and Ptch1 mRNA decrease level). Characteristically, molecular monitoring highlighted that all patients developing resistance to TKIs treatment, showed a tendency to Hh activation (high and low mRNA levels of Patch1 and Smo respectively) few months before the development of Abl KD mutation (10/10). Typically this last behaviour was more evident in patients developing the gatekeeper mutation T315I. Finally, in accordance with published data, we noted that the pharmacological inhibition of Hh signalling impairs the growth of TKIs resistant human CML cell line BaF/T315I, suggesting a novel treatment option. All our data provide molecular evidence for a role of the stemness pathway to predict quickly both the relapse and the TKIs resistance during CML treatment. Therefore, we propose to use Ptch1 and Smo mRNA levels together with BCR-ABL for the molecular monitoring of CP CML.