Hedgehog Pathway Mutations Drive Oncogenic Transformation in High-Risk T-Cell Acute Lymphoblastic Leukemia

Despite recent advances in improving outcome for patients with T-cell acute lymphoblastic leukemia (T-ALL), those who relapse or who have resistant disease are in urgent need of new therapeutic approaches. Recently, mutations in the Hedgehog pathway have been described in T-ALL, but the role of Hedgehog signaling in normal and malignant T-cell development is controversial, and whether these mutations are prognostic or drive oncogenic transformation is unknown, hindering the rationale for therapeutic intervention.

Through targeted next generation sequencing on a cohort of 109 diagnostic lymphoblast specimens from children with newly diagnosed T-ALL enrolled on contemporary clinical trials (DFCI 05-001 and COG AALL0434), we identified mutations in the Hedgehog pathway in 16% of patients (n = 17/109); PTCH1, the major negative regulator of the pathway, was the most frequently mutated Hedgehog pathway gene with 8% of patients harboring heterozygous missense mutations (n = 9/109). Remission bone marrow samples were evaluable by Sanger sequencing for 5 of the 9 PTCH1 mutations, and revealed that 3 of these mutations were absent at remission, indicating a somatic origin, whereas, 2 of these mutations were detected at remission, suggestive of either an underlying cancer predisposition syndrome, or somatic mutations associated with clonal hematopoiesis. Hedgehog pathway mutations predicted resistance to induction chemotherapy, as defined by ≥ 5% residual lymphoblasts in the bone marrow by morphology or flow cytometry (P= 0.009).

To investigate the role of Hedgehog signaling in T-ALL, we focused on PTCH1 because this was the most commonly mutated Hedgehog gene identified. Transduction of wild-type PTCH1 into a PTCH1 -mutant T-ALL cell line downregulated Hedgehog pathway activity, impaired viability (P < 0.001), and induced apoptosis (P= 0.005). The effect on both viability and apoptosis was reversed by downstream Hedgehog pathway activation using a small molecule Smoothened agonist (P < 0.001 and P= 0.007, respectively), indicating on-target toxicity. Most of the PTCH1 mutations identified in human T-ALL were significantly less toxic than wild-type PTCH1 when transduced into PTCH1 -mutant T-ALL cells, indicating these encode functionally defective alleles.

To test whether PTCH1 mutations accelerate the onset of T-ALL in vivo, we turned to a CRISPR/Cas9 system for tissue-specific gene disruption in transgenic zebrafish. We first identified guide RNAs that effectively mutagenized zebrafish ptch1, or the locus syntenic to the human AAVS1 safe-harbor locus as a negative control. Guide RNAs were cloned into a vector that drives ubiquitous expression of the guide RNA, together with lineage-restricted expression of a Cas9-T2A-GFP self-cleaving protein under the control of the zebrafish rag2 promoter. Using this model, we found that inducing ptch1 mutations in thymocytesaccelerated the onset of notch1- induced T-ALL, with a median time to T-ALL onset of 12.3 weeks in the ptch1 -mutant group versus median not reached for the aavs1 -control group (P= 0.0004). These ptch1 -mutant leukemias were transplantable into irradiated wild-type recipients, and subsequent treatment of these animals with the Hedgehog pathway inhibitor cyclopamine had significant therapeutic activity (P= 0.002).

In conclusion, our findings indicate that Hedgehog-activating mutations are associated with resistance to induction chemotherapy and drive oncogenic transformation in high-risk T-ALL. Together, these findings provide a molecular rationale for targeted therapy with Hedgehog pathway inhibitors for patients with PTCH1 -mutant high-risk T-ALL.