Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics.

Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

The association between cannabis use and outcome in pharmacological treatment for opioid use disorder.

BACKGROUND: With the ongoing opioid crisis and policy changes regarding legalization of cannabis occurring around the world, it is necessary to consider cannabis use in the context of opioid use disorder (OUD) and its treatment. We aimed to examine (1) past-month cannabis use in patients with OUD, (2) self-reported cannabis-related side effects and craving, and (3) the association between specific characteristics of cannabis use and opioid use during treatment in cannabis users. METHODS: Participants receiving pharmacological treatment for OUD (n = 2315) were recruited from community-based addiction treatment clinics in Ontario, Canada, and provided information on past-month cannabis use (self-report). Participants were followed for 3 months with routine urine drug screens in order to assess opioid use during treatment. We used logistic regression analysis to explore (1) the association between any cannabis use and opioid use during treatment, and (2) amongst cannabis-users, specific cannabis use characteristics associated with opioid use. Qualitative methods were used to examine responses to the question: "What effect does marijuana have on your treatment?". RESULTS: Past-month cannabis use was reported by 51% of participants (n = 1178). Any cannabis use compared to non-use was not associated with opioid use (OR = 1.03, 95% CI 0.87-1.23, p = 0.703). Amongst cannabis users, nearly 70% reported daily use, and half reported experiencing cannabis-related side effects, with the most common side effects being slower thought process (26.2%) and lack of motivation (17.3%). For cannabis users, daily cannabis use was associated with lower odds of opioid use, when compared  with occasional use (OR = 0.61, 95% CI 0.47-0.79, p < 0.001) as was older age of onset of cannabis use (OR = 0.97, 95% CI 0.94, 0.99, p = 0.032), and reporting cannabis-related side effects (OR = 0.67, 95% CI 0.51, 0.85, p = 0.001). Altogether, 75% of cannabis users perceived no impact of cannabis on their OUD treatment. CONCLUSION: Past-month cannabis use was not associated with more or less opioid use during treatment. For patients who use cannabis, we identified specific characteristics of cannabis use associated with differential outcomes. Further examination of characteristics and patterns of cannabis use is warranted and may inform more tailored assessments and treatment recommendations.

Are patients' goals in treatment associated with expected treatment outcomes? Findings from a mixed-methods study on outpatient pharmacological treatment for opioid use disorder.

OBJECTIVES: Existing methods of measuring effectiveness of pharmacological treatment for opioid use disorder (OUD) are highly variable. Therefore, understanding patients' treatment goals is an integral part of patient-centred care. Our objective is to explore whether patients' treatment goals align with a frequently used clinical outcome, opioid abstinence. DESIGN: Triangulation mixed-methods design. SETTING AND PARTICIPANTS: We collected prospective data from 2030 participants who were receiving methadone or buprenorphine-naloxone treatment for a diagnosis of OUD in order to meet study inclusion criteria. Participants were recruited from 45 centrally-managed outpatient opioid agonist therapy clinics in Ontario, Canada. At study entry, we asked, 'What are your goals in treatment?' and used NVivo software to identify common themes. PRIMARY OUTCOME MEASURE: Urine drug screens (UDS) were collected for 3 months post-study enrolment in order to identify abstinence versus ongoing opioid use (mean number of UDS over 3 months=12.6, SD=5.3). We used logistic regression to examine the association between treatment goals and opioid abstinence. RESULTS: Participants had a mean age of 39.2 years (SD=10.7), 44% were women and median duration in treatment was 2.6 years (IQR 5.2). Six overarching goals were identified from patient responses, including 'stop or taper off of treatment' (68%), 'stay or get clean' (37%) and 'live a normal life' (14%). Participants reporting the goal 'stay or get clean' had lower odds of abstinence at 3 months than those who did not report this goal (OR=0.73, 95% CI 0.59 to 0.91, p=0.005). Although the majority of patients wanted to taper off or stop medication, this goal was not associated with opioid abstinence, nor were any of their other goals. CONCLUSIONS: Patient goals in OUD treatment do not appear to be associated with programme measures of outcome (ie, abstinence from opioids). Future studies are needed to examine outcomes related to patient-reported treatment goals found in our study; pain management, employment, and stopping/tapering treatment should all be explored.