7019 Background: Midostaurin is approved for FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML), however efficacy has also been observed in a subpopulation of FLT3 mutant-negative AML, suggesting that FLT3 mutation is not the only determinant in conferring midostaurin sensitivity. We previously described a phosphoproteomic signature significantly elevated in primary AML blasts that responded to midostaurin ex vivo (Casado et al Leukaemia 2018). This signature includes phosphorylation sites on protein kinase C delta (a midostaurin off-target) and its substrate GSK3A. In this study, we tested whether these phospho-signatures could group FLT3+ patients based on clinical responses to midostaurin plus chemotherapy. Methods: We obtained FLT3+ bone marrow (BM) and peripheral blood (PB) diagnosis specimens (n=56 cases) from the Leukemia Tissue Bank at Princess Margaret Cancer Centre. These patients were treated with standard chemotherapy plus midostaurin. Phospho-signatures quantified using mass spectrometry were analysed with a classification machine learning algorithm to group patients based on response to treatment as a function of phospho-signature status. Other features (e.g. genetic mutations, HSC-transplant) were also analysed. Differential survival analysis was carried out with Kaplan-Meier and Log Rank test methods. Phospho-signatures for BM and PB samples were analysed independently. Results: A first ML model was developed based on the signature described in the Casado et al study. Patients positive for this signature exhibited a survival probability of 243 weeks, compared to 126 weeks in signature negative patients (averages by geometric mean, Log Rank p = 9.88e-05). As the patients in the current study received chemotherapy, in addition to midostaurin, we also identified a new signature consisting of 26 phospho-sites (model 2), which partially overlapped with the first model. Patients positive for model 2 signature showed a markedly longer survival time than negative patients (269 vs 76 weeks, Log Rank p = 1.30e-05 for PB and 241 vs 56, Log Rank p = 2.13e-09 for BM specimens, Table). No other features separated survival as clearly as model 2. Conclusions: We have identified phospho-signatures with the potential to further stratify FLT3+ AML for midostaurin treatment. The presence of PRKCD signalling components in signatures provides a rationale for midostaurin activity in sensitive cases. Analysis will also be performed on FLT3 mutant-negative cases to validate the signature in this group.[Table: see text]
Abstract Background: NSCLC cells carrying EGFR mutations can gain resistance to cognate TKIs through amplification of Chr22q11.2 (Chr22amp), a chromosome segment containing CRKL. This also specifically associates with exquisite sensitivity to inhibitors of Aurora Kinase B (AZD2811), potentially mediated by other Chr22 genes. Furthermore, a phenotypic rewiring occurs in the response to AZD2811, from a senescent polyploidy in wildtype (WT) cells to apoptosis in Chr22amp cells. Here, we aimed to elucidate the underlying signaling alterations in this background by phosphoproteomic pathway analysis. Methods: The EGFR mutant cell line PC9 and 8 TKI resistant derivatives were profiled (4 Chr22amp and 4 WT). Kinetics of response to AZD2811 (100nM) and osimertinib (160 nM) were identified by flow cytometry. Samples (n=3) were prepared for phosphoproteomics, after 6, 24, and 48 h AZD2811 and 1 h osimertinib, with time matched controls. Cells were washed and lysed in urea, then digested with trypsin. Phosphorylated peptides were enriched with TiO2 and analyzed by Orbitrap LC-MS/MS. Computational analyses quantified peptides across samples. KScanTM bioinformatics identified differential phosphopeptides between Chr22amp and WT to determine kinase substrate profiles by KSEA, putative downstream targets (PDT) and differential compound target activity markers (CTAM). Results: Single cell time-course analysis of phenotypic response to AZD2811 in Chr22amp cells showed that >60% of cells become Annexin V+ by 48 h post-treatment. We took earlier timepoints of 6, 24 and 48 h post treatment. We focused the phosphoproteomic analysis on three comparisons of Chr22amp amplified cells to: 1) the basal signaling state compared to WT; 2) the signaling response to osimertinib in parental PC9; and 3) the altered kinetics of signaling in response to AZD2811 compared to WT. At the basal level, Chr22amp had CK1e, CDK2, p38a substrates differentially enriched, and MTOR inhibitor and Aurora B inhibitor modulated sites (p<10-3). The response to osimertinib was largely differential in the maintenance of ERK1/2 signaling to P90RSK1 but not MEK1 in Chr22amp cells. In cells treated with AZD2811, alterations in signaling were associated with Aurora B in all cells as expected. However in amplified cells, we observed key differences at 24h such in cell death and metabolic processes in specific hierarchical clusters of temporally modulated sites, underpinned by relative down regulation of multiple signaling nodes such as ARAF (z = 4.87, p<10-2), ERN1 (z = 4.56, p<10-2), and CDK2 (z = 4.30, p<10-2). Conclusions: Here, we identified significant pathway deregulation in Chr22amp cells that subverted EGFR inhibition and enhanced sensitivity to AZD2811. Intriguingly, we detected enhanced Aurora B activity in Chr22amp cells at basal levels, and surprising impact of AZD2811 on the EGFR pathway. Citation Format: Arran Dokal, Jordi Bertran-Alamillo, Edmund Wilkes, Hilary Lewis, Ana Gimenez-Capitan, Calum Greenhalgh, Ruth Osuntola, Maruan Higazi-Vega, Shona Ellison, Vinothini Rajeeve, Giulia Fabbri, Urszula Polanska, J. Elizabeth Pease, Pedro Rodriguez-Cutillas, Jelena Urosevic, Miguel Angel Molina-Vila, David Britton, Jon Travers. Precision phosphoproteomic analysis in Chr22q11.2 amplified NSCLC cells reveals distinct signaling corruption and response to Aurora kinase B inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1107.
Abstract Background: Midostaurin (mido) is approved for treatment of FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML). However, FLT3 mutation is not the only determinant of mido sensitivity. Here we report phosphoprotein signatures which predict response to chemotherapy (chemo) plus mido, and identify active drug targets on potential resistance pathways. Methods: Samples collected at diagnosis, post-treatment and relapse from FLT3+ patients treated with chemo+mido were obtained from the Leukemia Tissue Bank at the Princess Margaret Cancer Centre. Peptides and enriched phosphopeptides from bone marrow (BM) and peripheral blood (PB) mononuclear cells were quantified using liquid chromatography-tandem mass spectrometry. Signatures for BM/PB diagnosis samples were analyzed independently and used to train a classification machine learning algorithm to group patients (n=54) based on response to treatment. Additional features (e.g. genetic mutations) were also analyzed. Kaplan-Meier and Log-Rank test methods were used to assess differential survival between patient groups. To investigate pathways potentially driving resistance to chemo+mido, differential protein phosphorylation indexes were identified through comparison of post-treatment or relapse samples to paired diagnosis samples. To account for population heterogeneity, a filter was applied based on frequency of observation. Activated pathways potentially driving resistance were identified with functional enrichment tools and kinase-substrate enrichment analysis. Statistical significance of enrichment were determined using parametric methods and p-values adjusted for multiple testing using the Benjamini-Hochberg method. Results: Patients positive for a signature consisting of 26 phosphorylation sites showed a markedly longer survival time than negative patients (PB: 269 vs 76 weeks, Log-Rank p=1.30e-05; BM: 241 vs 56, Log-Rank p=2.13e-09). This signature partially overlapped with an ex-vivo signature of response to mido, described previously by Casado et al (Leukemia, 2018). A proteomic signature was also identified, with positive patients showing a longer survival time than negative patients (PB: 330 vs 173 weeks, Log-Rank p=5.0e-04; BM: 460 vs 156, Log-Rank p=5.2e-06). Key, diverging phosphorylation site signatures were identified between patients with refractory disease/early relapse and patients with complete response and no relapse or death within 2 years post-treatment. Pathways with increased activity in post-treatment or relapse specimens were associated with molecular functions such as regulation of cell proliferation, migration, differentiation and anti-apoptosis. Conclusions: We identified phosphoproteomic and proteomic signatures that differentiate survival mediated by response to chemo+mido. While the former was more predictive, both may enable further stratification of FLT3+ AML receiving mido treatment. Drug targets on pathways demonstrating increased activity in relapse/refractory cases may play a role in resistance; this will be determined in follow-up inhibitor studies. Citation Format: Luis Veiga Nobre, Celia Colomina Basanta, Salvatore Federico Pedicona, Arran David Dokal, Andrea Arruda, Ryan Smith, Calum Greenhalgh, Francesca Patella, Pedro Maria Casado-Izquierdo, Bela Wrench, Jane Theaker, Andrew Thompson, Mark D. Minden, John G. Gribben, David James Britton, Pedro Rodriguez Cutillas. Phosphoproteomics reveals active drug targets on pathways of resistance and predicts response to midostaurin plus chemotherapy in FLT3 mutant-positive acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA013.
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