Background: Dysregulation of the cyclin D-CDK4/6-Rb axis occurs in a substantial proportion of ER-positive (ER+) breast cancers and has been linked with endocrine resistance. Adding the CDK4/6 inhibitor palbociclib to endocrine treatment has led to a substantial improvement of the outcome of patients with ER+ metastatic breast cancer. However, with the increasing clinical use, acquired resistance to palbociclib is merging as a new major clinical challenge. Methods: The ER+ cell lines T47D and MCF7 have been shown to be highly sensitive to treatment with palbociclib. Using long-term co-culture with increasing doses of Palbociclib, we generated MCF7 and T47D cell line clones with acquired resistance to Palbociclib. Three distinct resistant clones were selected for each cell line showing an IC50 shift from sensitive to resistant of approximately 300nM to 3uM for MCF7 and 400nM to 3.5uM for T47D, respectively. Resistant cell lines were characterized using RNA sequencing and mass spectrometry-based phosphoproteomics. Effects on selected target proteins (eg pAKT, pS6, pRB, RB or Cyclin D1) were confirmed using Western Blots. To modify resistance to palbociclib, a targeted in vitro drug-screen was performed using a range of inhibitors of the PI3K/AKT/mTOR and MEK pathways. Results: Western blot analysis of resistant cell lines demonstrated sustained down-regulation of Rb and phospho-Rb in response to palbociclib, which was reversible after discontinuation of palbociclib. Mass spectrometry identified >6,000 peptides across parental and resistant cells corresponding to 4,757 phospho-peptides and 5,337 phosphorylation sites. Pathway analysis suggested increased activity in the P3IK/AKT/mTOR pathway in resistant clones (including Akt1, p90S6K and mTOR), as well as changes in p53 and apoptotic regulation (e.g. phosphorylation of BAD). In addition, resistant clones showed multiple phosphorylation changes in the Rho/Rac pathway, suggesting changes in cytoskeletal organisation and a more invasive phenotype. Targeted drug screening showed a variable pattern across resistant clones with increased sensitivity to co-treatment of palbociclib with AKT inhibitors, PI3K alpha/delta inhibitors and/or MEK inhibitors in selected resistant clones, whereas pan-PI3K or PI3K beta/delta inhibitors showed limited efficacy in the selected clones. Conclusions: Phosphoproteomic analysis of palbociclib-resistant ER+ breast cancer cell lines demonstrated up-regulation of PI3K/AKT/mTOR and anti-apoptotic pathways. Resistant cell lines were sensitive to inhibition of PI3K/AKT/mTOR and/or MEK pathways with distinct patterns of activity across resistant clones suggesting that co-treatment of CDK4/6 inhibitors and PI3K/AKT and/or MEK inhibitors warrants further investigation as potential new therapeutic strategies in palbociclib resistance. Citation Format: Lenihan C, Bouchekioua-Bouzaghou K, Shia A, Wilkes E, Casado-Izquierdo1 P, Cutillas P, Schmid P. Characterization of resistance to the selective CDK4/6 inhibitor palbociclib in ER positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-02.

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Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Background: Dysregulation of the cyclin D-CDK4/6-Rb axis occurs in a substantial proportion of ER-positive (ER+) breast cancers promoting proliferation and resistance. Adding the CDK4/6 inhibitor palbociclib to endocrine treatment has led to a substantial improvement of the outcome of patients with ER+ metastatic breast cancer. However, with the expected clinical implementation of CDK4/6 inhibitors, acquired resistance is emerging as a major clinical challenge. Using long-term culture with palbociclib, we have established MCF7 and T47D cell lines with 8-10 fold increased IC50 values as a model of acquired resistance. Characterization of resistant cell lines using phosphoproteomic analysis and RNAseq revealed increased activity in the PI3K/AKT/mTOR pathway which can be targeted therapeutically by co-treatment with CDK4/6 inhibitors and PI3K/AKT/mTOR inhibitors (Lenihan C, et al, San Antonio Breast Cancer Symposium 2015). The present work addresses whether palbociclib still has target effects and/or residual activity in resistant cells, to characterize cell cycle pathways in resistant cells, to establish the role of ER-signaling in resistant cells, and to study if palbociclib resistance might be associated with a more aggressive phenotype. Results: Western blot analysis of newly generated resistant clones demonstrated significant reduction of phospho RB (ser780), in keeping with maintained target inhibition with palbociclib. Although total RB decreased in some clones, the protein remained detectable in all resistant clones. Cumulative population doubling confirmed palbociclib still has an effect on cell proliferation in resistant clones. Subsequent long-term culture in the presence of palbociclib altered the effect of the drug; these cells gradually lose the inhibition of pRB, as well as the residual anti-proliferative effect of palbociclib, suggesting potential loss of palbociclib target effects. Mutational analysis of RB is ongoing. Cell cycle analysis of resistant clones demonstrated an increase in cyclin E expression suggesting up-regulation of alternative cell cycle pathways. Morphological changes of resistant clones and multiple phosphorylation changes in the Rho/Rac pathway showed by our phosphoproteomic analysis suggested that resistant clones might exhibit a more aggressive phenotype. Tumor spheroid-based invasion assays and transwell migration assays confirmed increased migration and invasion of resistant clones compared to sensitive cells. Conclusions: Whilst Palbociclib has sustained target effects and anti-proliferative activity during early stages of resistance, these effects gradually diminish during later stages of acquired resistance. Palbociclib resistant breast cancer cells also exhibit a more aggressive phenotype. In vivo investigation is ongoing to better characterize the escape mechanisms involved in CDK4/6 inhibitors resistance. Citation Format: Katia Bouchekioua-Bouzaghou, Catherine Lenihan, Alice Shia, Edmund Wilkes, Pedro Casado-Izquierdo, Pedro Cutillas, Peter Schmid. Characterization of the mechanisms of early and later stages of resistance to the selective CDK4/6 inhibitor palbociclib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2819.

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An early event in lung oncogenesis is loss of the tumour suppressor gene LIMD1 (LIM domains containing 1); this encodes a scaffold protein, which suppresses tumourigenesis via a number of different mechanisms. Approximately 45% of non-small cell lung cancers (NSCLC) are deficient in LIMD11, yet this subtype of NSCLC has been overlooked in preclinical and clinical investigations. Defining therapeutic targets in these LIMD1 loss-of-function patients is difficult due to a lack of druggable targets, thus alternative approaches are required. To this end, we performed the first drug repurposing screen to identify compounds that confer synthetic lethality with LIMD1 loss in NSCLC cells. PF-477736 was shown to selectively target LIMD1 deficient cells in vitro through inhibition of multiple kinases, inducing cell death via apoptosis. Furthermore, PF-477736 was effective in treating LIMD1-/- tumours in subcutaneous xenograft models, with no significant effect in LIMD1+/+ cells. We have identified a novel drug tool with significant preclinical characterization that serves as an excellent candidate to explore and define LIMD1-deficient cancers as a new therapeutic subgroup of critical unmet need.

Human chromosome translocations at 11q23, disrupting the MLL1 gene, result in poor prognostic mixed lineage leukaemias. Current chemotherapy treatment protocols produce an unsatisfactory outcome. Indeed, the average five-year event free survival rate is 44% in paediatric cases, and adult cases have been estimated as low as 15% for two-year survival rates, indicating there is an unmet critical need for more effective therapies. In recent years, there has been great interest in targeting the epigenetic factors involved in MLL-rearranged (MLL-r) leukaemic transformation and maintenance; however, epigenetic plasticity, the potential role of the remaining MLL1 allele and the elusive leukaemic stem cells present in acute myeloid leukaemia (AML), provide many routes to chemoresistance. There is currently great interest in targeting the cell cycle and key intracellular signalling pathways (e.g. Wnt signalling), independent of specific aberrant lesions in AML (e.g. MLL-fusion proteins, DNMT3a mutants), to combat highly quiescent leukaemic stem cells, which are the most difficult to eradicate. In addition, protection of the resident normal haematopoietic stem cells (HSCs), during aggressive induction chemotherapy protocols, provides another route to reduce the competitive advantage of AML cells in vivo. We previously identified two new genes, involved in the regulation of MLL1, Wnt signalling and the cell cycle: the CDK subunits CKS1 and CKS2 (Grey et al. 2017). Here, we investigated the roles of CKS1 and CKS2 during normal and malignant haematopoiesis in vivo, revealing differences in key signalling pathways involved in haematopoiesis and leukaemogenesis, implicating the CKS1/CKS2 axis as a valid therapeutic target. We demonstrate that primary AML patient samples, engrafted in immune deficient mice, are sensitive to inhibition of CKS1-dependent protein degradation, with reduced tumour burden after treatment and significant improvement in survival times. In addition, patient samples showed CKS1-sensitivity irrespective of inherent resistance to Cytarabine. Current chemotherapy protocols, using Cytarabine and Doxorubicin, can be significantly deleterious to resident normal HSCs in vivo. Transient inhibition of CKS1-dependent protein degradation, in vivo, provides a protective function to human CD34+ HSPCs when treated with Cytarabine/Doxorubicin (5+3 dosing protocol), resulting in reduced apoptosis and increased stem cell potential post-therapy. Importantly, combination treatment of CKS1 inhibition with Cytarabine/Doxorubicin significantly reduces AML tumour burden and improves overall survival, by selectively killing AML cells and preserving normal resident HSCs. Altogether, these results open a promising alternative approach for modulating protein phosphorylation and degradation to selectively target leukaemic cells, with the great advantage to protect normal resident HSCs from cytotoxic effects of induction chemotherapy. Disclosures No relevant conflicts of interest to declare.

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]