Abstract Philadelphia-positive acute lymhoblastic leukemia (Ph+ ALL) and Ph1-like B-ALL account for most cases of “high-risk” adult B-ALL. Current therapies with tyrosine kinase inhibitors (TKIs) have improved the outcome of Ph+ ALL, but resistance to TKIs develops rapidly in most patients. Ph1-like B-ALL is currently treated with intensive combination chemotherapy but disease relapse is common with a 5-year survival in only ~25% of patients. As a result, the prognosis of Ph+ ALL and Ph1-like B-ALL remains dismal. In previous studies, we showed that Ph+ and Ph1-like ALL cells exhibit a selective requirement for CDK6 expression while CDK4 expression is dispensable. CDK6 is the catalytic subunit of the cyclin D/CDK6 complex which is essential for the G1 to S-phase cell cycle transition and has kinase-independent growth-promoting effects in hematological malignancies. Our preliminary studies indicate that CDK6 silencing is more effective than CDK6 enzymatic inhibition in suppressing Ph+ ALL in mice. To block kinase-dependent and independent effects of CDK6, we have developed CDK4/6-targeted proteolysis-targeting chimera (PROTACs) that inhibit CDK4/6 enzymatic activity in vitro and promote the preferential degradation of CDK6 over CDK4 in Ph+ and Ph1-like ALL cells, providing durable suppression of CDK6 function. In this proposal, we will assess the requirement of CDK6 in Ph+ and Ph1-like ALL by comparing the effects of CDK6 degradation by PROTAC YX-2-107 and pharmacological inhibition using Palbociclib, an FDA-approved CDK4/6 inhibitor (Aim 1.1). We will also determine whether the more potent leukemia suppression induced by CDK6 down-regulation in comparison to CDK6 enzymatic inhibition can be explained by changes in gene expression induced selectively by CDK6 silencing. Such changes involve the histone deacetylase 1(HDAC1) gene and several others involved in mitochondrial metabolic pathways (Aims 1.2 and 1.3). Although we have been able to achieve high specificity of CDK6 versus CDK4 targeting and biological/therapeutic effects comparable/superior to Palbociclib ex vivo and in PDXs of Ph+ ALL, we will continue to improve our lead compound PROTAC YX-2-107 by medicinal chemistry approaches in order to develop derivatives with enhanced in vivo efficacy. In Aim 2, we will assess metabolic properties of select CDK6-degrading PROTACs and test their biological/therapeutic effects in Ph+ and Ph1-like ALL cells ex vivo and in mice injected with de novo or relapsed/TKI-resistant patient-derived Ph+/Ph1-like ALL cells. Collectively, our PROTAC-based approach which leverages the expertise in cancer biology and medicinal chemistry of the Calabretta and Salvino's laboratories holds promise to develop novel and more effective therapeutic agents for the treatment of CDK6-dependent high-risk B-ALL in pre-clinical PDX models and, potentially, in the clinic.