A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

Discovery of New Coumarin-Based Lead with Potential Anticancer, CDK4 Inhibition and Selective Radiotheranostic Effect: Synthesis, 2D & 3D QSAR, Molecular Dynamics, In Vitro Cytotoxicity, Radioiodination, and Biodistribution Studies

Novel 6-bromo-coumarin-ethylidene-hydrazonyl-thiazolyl and 6-bromo-coumarin-thiazolyl-based derivatives were synthesized. A quantitative structure activity relationship (QSAR) model with high predictive power r2 = 0.92, and RMSE = 0.44 predicted five compounds; 2b, 3b, 5a, 9a and 9i to have potential anticancer activities. Compound 2b achieved the best ΔG of –15.34 kcal/mol with an affinity of 40.05 pki. In a molecular dynamic study 2b showed an equilibrium at 0.8 Å after 3.5 ns, while flavopiridol did so at 0.5 Å after the same time (3.5 ns). 2b showed an IC50 of 0.0136 µM, 0.015 µM, and 0.054 µM against MCF-7, A-549, and CHO-K1 cell lines, respectively. The CDK4 enzyme assay revealed the significant CDK4 inhibitory activity of compound 2b with IC50 of 0.036 µM. The selectivity of the newly discovered lead compound 2b toward localization in tumor cells was confirmed by a radioiodination biological assay that was done via electrophilic substitution reaction utilizing the oxidative effect of chloramine-t. 131I-2b showed good in vitro stability up to 4 h. In solid tumor bearing mice, the values of tumor uptake reached a height of 5.97 ± 0.82%ID/g at 60 min p.i. 131I-2b can be considered as a selective radiotheranostic agent for solid tumors with promising anticancer activity.

Tankyrase inhibition sensitizes cells to CDK4 blockade

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the β-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.

Identification of a novel, NF-κB nucleolar stress response pathway

Abstractp53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-κB transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-κB also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-κB pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-κB signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-κB induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-κB pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-κB pathway. Finally, usingex vivoculture, we show a strong correlation between degradation of TIF-IA and activation of NF-κB in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-κB nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

PIK3IP1 Inhibition of PI3K in G1 Arrest Induced By CDK4 Inhibition Reprograms MCL for Ibrutinib Therapy

Inhibition of CDK4/6 has emerged as a promising therapy for diverse human cancers. Mantle cell lymphoma (MCL), in which aberrant cyclin D1 expression and CDK4 activity underlie unrestrained proliferation of tumor cells, remains incurable. Targeting Bruton tyrosine kinase (BTK) with ibrutinibin recurrent MCLhas unprecedented single agent activity, with a completion remission (CR) rate of 21%. However, progression on ibrutinib is virtually universal in MCL and is associated with aggressive proliferation of tumor cells and a dismal clinical outcome. By longitudinal integrated RNA- and exome-seq analysis, we previously discovered a mutation in the ibrutinib binding site (BTKC481S) in MCL that was specific to relapse from a durable response, but absent in both primary resistance and resistance following a transient response. Irrespective of mechanism, ibrutinib resistance was associated with enhanced PI3K activation as determined by Western blotting of MCL cells isolated from serial biopsies. Induction of prolonged early G1 arrest (pG1) by selective inhibition of CDK4 with palbociclib sensitized to PI3K inhibition independent of this BTK mutation and to ibrutinib when the wild type BTK was retained. In both cases, synergistic killing appears to occur via cooperative inactivation of PI3K. Collectively, our data suggest that dual inhibition of CDK4 and BTK or PI3K restrain the expansion of resistant clones or even eliminate them to deepen or prolong the clinical response to targeting BTK. To test this hypothesis, we initiated a phase I clinical trial of palbociclib plus ibrutinib in recurrent MCL patients in August of 2014. All 5 dose levels included ibrutinib daily and palbociclib administered on days 1-21 of each 28-day cycle (Figure 1). Based on an intent-to-treat analysis of 18 patients, 12 (67%) patients have responded to treatment including 8 (44%) complete responses (CR). The median time to CR was 3 cycles and no responding patients have progressed on study. The therapy was well tolerated (see ASH abstract by Martin et al for clinical information). RNA- and exome-seq were performed to assess the genomic alternations and gene expression on purified MCL cells from samples before therapy, on progression and on treatment from PR patients. Initial analysis did not detect non-synonymous mutation in CDK4, CCND1, or genes in the BCR or PI3K signaling pathway. Hemizygous deletion of Rb, p53, ATM and the INK4A/B locus and amplification of PIK3CA and PIK3CB occurred in 30-50 % of patients before therapy. These copy number variations were predominantly associated with primary resistance (4 subjects) but did not preclude CR or PR. By contrast, expression of PIK3IP1, a putative PI3K inhibitor, was markedly reduced on progression in all primary resistant patients, but not in the PR patient. Function studies demonstrated that PIK3IP1 was required for pG1 sensitization to BTK and PI3K inhibition in MCL cells in vitro. Moreover, PIK3IP1 was induced in pG1 together PI3K inhibition in MCL cells in patients responding to palbociclib alone in a separate clinical trial (Martin, DiLiberto et al, unpublished), suggesting that PIK3IP1 mediates pG1 reprogramming. In summary, the high CR rate and durability support our hypothesis that induction of prolonged early G1 arrest by CDK4 inhibition reprograms MCL cells for deepened and durable targeting of BTK in MCL. Our data further suggest inhibition of PI3K by PIK3IP1 as a potential mechanism to overcome ibrutinib resistance. A phase II multi-center clinical trial is planned to further elucidate the genes and mechanisms that discriminate sensitivity from resistance to dual targeting of CDK4 and BTK. Figure 1. Dual targeting of CDK4 with palbociclib and BTK with ibrutinib in recurrent MCL Figure 1. Dual targeting of CDK4 with palbociclib and BTK with ibrutinib in recurrent MCL Disclosures Blum: Pharmacyclics: Research Funding. Ruan:Pharmacyclics: Research Funding, Speakers Bureau. Martin:Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Other: travel, accommodations, expenses; Novartis: Consultancy; Acerta: Consultancy; Teva: Research Funding.