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Mutations in the tyrosine kinase domain of the Anaplastic Lymphoma Kinase (ALK) oncogene in neuroblastoma occur most frequently at one of three hotspot amino acid residues, with the F1174* and F1245* variants conferring de novo resistance to first and second generation ALK inhibitors including crizotinib and ceritinib. Lorlatinib, a third generation ALK/ROS inhibitor, overcomes de novo resistance and induces complete and sustained tumor regressions in patient-derived xenograft (PDX) models unresponsive to crizotinib. Lorlatinib has now completed Phase 1 testing in children and adults with relapsed/refractory ALK-driven neuroblastoma and entered pivotal Phase 3 testing within the Children's Oncology Group. To define mechanisms underlying the superior activity of lorlatinib, we utilized a chemical proteomics approach to quantitatively measure functional kinome dynamics in response to lorlatinib and crizotinib in clinically relevant ALK-driven neuroblastoma PDX models. Lorlatinib was a markedly more potent inhibitor of ALK and preferentially downregulated several kinases implicated in G2/M cell cycle transition compared to crizotinib. Lorlatinib treatment also led to the repression of MYCN expression and its occupancy at promoters of the same G2/M kinases. These data providing mechanistic insight into the superior efficacy of lorlatinib over crizotinib for the treatment of ALK-driven neuroblastoma.

Resistance to endocrine therapy (ET) is common in estrogen receptor (ER) positive breast cancer. Multiple studies have demonstrated that upregulation of MAPK signaling pathways contributes to ET resistance. Herein we show that vandetanib treatment enhances sensitivity to ET in ET-sensitive and -resistant ER+ breast cancer models. Using a CRISPR knockout model, we demonstrate that vandetanib effects are partially mediated by RET receptor tyrosine kinase. Vandetanib treatment alters the gene expression program of ER+ breast cancer cells resulting in a less proliferative and more estrogen responsive Luminal-A like character. Tyrosine kinase network reprogramming was assessed using multiplexed kinase inhibitor beads-mass spectrometry (MIB/MS) assay to identify adaptive resistance mechanisms to vandetanib treatment, which identified upregulation of HER2 activity. Co-treatment to inhibit HER2 with lapatinib enhanced sensitivity to vandetanib, demonstrating biologic activity of HER2 upregulation. Finally, we use our operating room-to-laboratory assay that measures drug response in individual primary tumor cells in short term cultures to demonstrate conserved gene expression changes, including increased HER2 activity signatures, in vandetanib treated cells, and identify features associated with vandetanib response. These results support future investigation of RET targeting strategies considering reprogrammed networks, such as activated HER2, in patients with ET resistant ER+ breast cancer.

The kinome is a dynamic system of kinases regulating signaling networks in cells and dysfunction of protein kinases contributes to many diseases. Regulation of the protein expression of kinases alters cellular responses to environmental changes and perturbations. We configured a library of 672 proteotypic peptides to quantify >300 kinases in a single LC-MS experiment using ten micrograms protein from human tissues including biopsies. This enables absolute quantitation of kinase protein abundance at attomole-femtomole expression levels, requiring no kinase enrichment and less than ten micrograms of starting protein from flash-frozen and formalin fixed paraffin embedded tissues. Breast cancer biopsies, organoids, and cell lines were analyzed using the SureQuant method, demonstrating the heterogeneity of kinase protein expression across and within breast cancer clinical subtypes. Kinome quantitation was coupled with nanoscale phosphoproteomics, providing a feasible method for novel clinical diagnosis and understanding of patient kinome responses to treatment.

Introduction: Hypertension may be common among market salespersons who are mostly physically inactive throughout the day. However, the burden of hypertension in this population remains unknown. In this study, we determined the prevalence of undiagnosed hypertension and associated factors among market salesperson in Kitgum central market, Kitgum district, Northern Uganda., Methods: A cross-sectional study, recruiting market salespersons aged 18 years or older without a prior diagnosis of hypertension or currently on anti-hypertensive therapy was conducted. A standardized questionnaire was administered, and body mass index (BMI) estimated. Hypertension was defined as two consistent measurements of systolic blood pressure ≥140mmHg and/or diastolic blood pressure ≥90mmHg measured 4 hours apart. Multivariable logistic regression analysis was performed to determine factors independently associated with undiagnosed hypertension. P<0.05 was considered statistically significant., Results: We enrolled 240 participants. The mean age was 39.4 ± 12.8 years. Most (83.3%, n=199) participants were female and urban dwellers (88.3%, n=212). The prevalence of undiagnosed hypertension was 16.7% (n=40). Of the 40 participants with hypertension, 16 (40%) were younger than 40 years. Factors associated with undiagnosed hypertension were, age >50 years (adjusted odds ratio (aOR): 7.0, 95% confidence interval (CI): 1.9-25.6, p=0.003), male gender (aOR: 4.2, 95CI: 1.5-11.1, p=0.005), alcohol consumption (aOR: 2.6, 95CI: 1.1-6.0, p=0.021), and being overweight (aOR: 3.6, 95CI: 1.5-8.8, p=0.005)., Conclusion: About one in six of market salespersons had undiagnosed hypertension, with a disproportionately high burden among those younger than 40 years. A larger multi-centric study is recommended to confirm our findings., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Kilama et al.)

Background: Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells., Methods: Stable isotope metabolic labeling, media creatine depletion, and Na + /K + -ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na + /K + -ATPase and creatine uptake., Results: Inhibition of the Na + /K + -ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na + /creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na + /K + -ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na + /K + -ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na + /K + -ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells., Conclusions: These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na + /K + -ATPase pump activity., General Significance: These studies identify kinase regulation of the Na + /K + -ATPase as pivotal in regulating creatine uptake and energy metabolism in cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Continuous exposure of a pancreatic cancer cell line MIA PaCa-2 (Mia S ) to gemcitabine resulted in the formation of a gemcitabine-resistant subline (Mia R ). In an effort to discover kinase inhibitors that inhibited Mia R growth, Mia R cells were exposed to kinase inhibitors (PKIS-1 library) in a 384-well screening format. Three compounds (UNC10112721A, UNC10112652A, and UNC10112793A) were identified that inhibited the growth of Mia R cells by more than 50% (at 50 nM). Two compounds (UNC10112721A and UNC10112652A) were classified as cyclin-dependent kinase (CDK) inhibitors, whereas UNC10112793A was reported to be a PLK inhibitor. Dose-response experiments supported the efficacy of these compounds to inhibit growth and increase apoptosis in 2D cultures of these cells. However, only UNC10112721A significantly inhibited the growth of 3D spheroids composed of Mia R cells and GFP-tagged cancer-associated fibroblasts. Multiplexed inhibitor bead (MIB)-mass spectrometry (MS) kinome competition experiments identified CDK9, CLK1-4, DYRK1A, and CSNK1 as major kinase targets for UNC10112721A in Mia R cells. Another CDK9 inhibitor (CDK-IN-2) replicated the growth inhibitory effects of UNC10112721A, whereas inhibitors against the CLK, DYRK, or CSNK1 kinases had no effect. In summary, these studies describe a coordinated approach to discover novel kinase inhibitors, evaluate their efficacy in 3D models, and define their specificity against the kinome.

Baculoviral IAP repeat containing 6 (BIRC6) is a member of the inhibitors of apoptosis proteins (IAPs), a family of functionally and structurally related proteins that inhibit apoptosis. BIRC6 has been implicated in drug resistance in several different human cancers, however mechanisms regulating BIRC6 have not been extensively explored. Our phosphoproteomic analysis of an imatinib-resistant chronic myelogenous leukemia (CML) cell line (MYL-R) identified increased amounts of a BIRC6 peptide phosphorylated at S480, S482, and S486 compared to imatinib-sensitive CML cells (MYL). Thus we investigated the role of BIRC6 in mediating imatinib resistance and compared it to the well-characterized anti-apoptotic protein, Mcl-1. Both BIRC6 and Mcl-1 were elevated in MYL-R compared to MYL cells. Lentiviral shRNA knockdown of BIRC6 in MYL-R cells increased imatinib-stimulated caspase activation and resulted in a ~20-25-fold increase in imatinib sensitivity, without affecting Mcl-1. Treating MYL-R cells with CDK9 inhibitors decreased BIRC6 mRNA, but not BIRC6 protein levels. By contrast, while CDK9 inhibitors reduced Mcl-1 mRNA and protein, they did not affect imatinib sensitivity. Since the Src family kinase Lyn is highly expressed and active in MYL-R cells, we tested the effects of Lyn inhibition on BIRC6 and Mcl-1. RNAi-mediated knockdown or inhibition of Lyn (dasatinib/ponatinib) reduced BIRC6 protein stability and increased caspase activation. Inhibition of Lyn also increased formation of an N-terminal BIRC6 fragment in parallel with reduced amount of the BIRC6 phosphopeptide, suggesting that Lyn may regulate BIRC6 phosphorylation and stability. In summary, our data show that BIRC6 stability is dependent on Lyn, and that BIRC6 mediates imatinib sensitivity independently of Mcl-1 or CDK9. Hence, BIRC6 may be a novel target for the treatment of drug-resistant CML where Mcl-1 or CDK9 inhibitors have failed.