Malignant transformation typically involves multiple genetic lesions whose combined activity gives rise to cancer1. Our analysis of 1,148 patient-derived B-cell leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway characteristic for the differentiation status of these transformed B cells. Specifically, we show here the JAK/STAT5 signaling pathway supports the developmental stage-specific expansion of pro-B ALL whereas the ERK-pathway that of pre-B ALL. Mutations that were not aligned with the central oncogenic driver would activate divergent pathways and subvert malignant transformation. Oncogenic lesions in B-ALL frequently mimic survival and proliferation signals downstream of cytokine receptors (through activation of STAT5)2-7 or the pre-B cell receptor (through activation of ERK)8-13. STAT5- (372 cases) and ERK- (386 cases) activating lesions were frequently found but only co-occurred in ~3% (37) of cases (P=2.2E-16). Single-cell mutation and phosphoprotein analyses revealed that even in these rare cases, oncogenic STAT5- or ERK-activation were mutually exclusive and segregated to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed malignant transformation. Conversely, Cre-mediated deletion of divergent pathway components triggered leukemia-initiation and accelerated development of fatal disease. Thus, persistence of divergent signaling pathways represents a powerful barrier to malignant transformation while convergence on one principal driver defines a key event during leukemia-initiation. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits strongly synergized with direct inhibition of the principal oncogenic driver. Hence, pharmacological reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses and to overcome drug-resistance. Current treatment approaches for drug-resistant cancer are focused on drug-combinations to suppress the central oncogenic driver and multiple alternative pathways14-17. Here, we introduce a concept based on inhibition of the principal driver combined with pharmacological reactivation of divergent pathways.

Signaling input from divergent pathways subverts malignant B-cell transformation