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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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.

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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.