Abstract: In spite of the dramatically improved outlook for patients with ER+ breast cancer, a subset of patients are intrinsically non-responsive to anti-hormonal therapy alone, and even the responders invariably acquire resistance to this treatment modality. In the setting of established hormone resistance, most of these patients will receive chemotherapy, with limited benefit and considerable impact on quality of life due to toxicity. Given the sheer numbers of patients affected (approximately two-thirds of all breast cancers are ER+), the unmet need for well-tolerated therapies that overcome resistance to anti-hormonal agents remains very high. Strikingly, the vast majority ( & gt;90%) of ER+ breast cancers express a functional retinoblastoma protein (Rb), a tumor suppressor that represents a critical regulator of the G1 to S phase transition in mammalian cells. In its hypo-phosphorylated state, Rb suppresses the expression of proteins that are essential for commitment to S-phase entry and progression through the remainder of the eukaryotic cell cycle. The G1 cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) which function in complexes with the D-type cyclins (collectively termed cyclin D) initiate the phosphorylation of Rb and override the repressive effects of Rb on cell-cycle progression. In ER-driven breast cancers, oncogenic signaling through the ER stimulates the cyclin D-cdk4/6 -dependent phosphorylation of Rb, and this proliferative stimulus is frequently augmented by amplification of cyclin D or loss of expression of the cyclin D-cdk4/6 inhibitor, p16 (encoded by the CDKN2A gene), two alterations that lead to elevated and dysregulated cyclin D-cdk4/6 activity. The requirement for cyclin D-cdk4/6 activity to circumvent the cell-cycle braking action of Rb suggested that ER+ breast cancer cells might be vulnerable to drug-induced inhibition of cdk4/6 activity. PD-0332991 is an orally administered, selective inhibitor of cyclin D-cdk4/6 kinase activities. In preclinical models, this drug showed significant anti-proliferative activity in Rb+ breast cancer cell lines, the majority of which also expressed the ER. The most well established substrate for the cyclin D-cdk4/6 is Rb, which is modified at multiple phosphoacceptor sites (including Ser-780, Ser-795, Ser-807, and Ser-811) by these cdks. The phosphorylation status of the Ser-807 and Ser-811 residues in Rb proved to be the most robust pharmacodynamic readouts for PD-0332991 activity in vivo, and serve as appropriate biomarkers for target modulation by this drug under in vitro and in vivo study conditions. The IC50 for reduction of Rb phosphorylation at these sites in two breast cancer cell lines (MCF7 and T47D) was approximately 20 nM. Similarly, we observed that PD-0332991 inhibits S-phase entry (measured by incorporation of thymidine into newly synthesized DNA) and cellular proliferation in panel of Rb+ cancer cell lines, with IC50 values ranging from 40-170 nM. The antiproliferative effects of PD-0332991 were not limited to transformed cells, reinforcing the idea that this drug was capable of suppressing the proliferation of any human cell type that requires cyclin D-cdk4/cdk6 activity to bypass the G1 restriction point imposed by Rb. Conversely, cancer cells that have inactivated Rb through genetic or epigenetic mechanisms are highly resistant (IC50 & gt; 3 uM) to the inhibitory effects of PD-0332991 on cell-cycle progression and cellular proliferation. Xenograft experiments confirmed the favorable in vivo safety profile and antitumor activity of PD-0332991. The maximum tolerated dose (MTD; defined as the dose that induced & lt;10% lethality) in SCID mice was 150 mg/kg/day when administered orally, once per day, for 14 days. Daily dosing at levels equal to or less than this MTD resulted in significant growth inhibition in many but not all Rb+ human tumor xenograft models. These studies in xenograft-bearing mice also confirmed that PD-0332991 was inactive at therapeutically obtainable concentrations against Rb-negative tumors. These preclinical studies provided a launching pad for a clinical trial in post-menopausal women with locally advanced or metastatic ER+ (HER2-) breast cancer. In this Phase 2 study, PD-0332991 was given in combination with letrozole (an inhibitor of estrogen synthesis), with the control arm receiving letrozole only. An interim analysis showed that the PD-0332991 plus letrozole combination yielded a dramatic improvement in treatment outcomes, more than tripling progression-free survival relative to the letrozole-only treatment arm. The drug was generally well-tolerated, due in part to its high degree of selectivity for cdk4/6 relative to other members of the cyclin-dependent kinase family, as well as other members of the extended kinase superfamily. These positive results led to a Breakthrough Therapy designation in 2013 for PD-0332991 in the treatment of patients with ER+ breast cancer. This presentation will provide information on the mechanisms of antitumor action of PD-0332991, together with updates on the current status of clinical development of this new addition to the armentarium of breast cancer treatments. Citation Format: Robert T. Abraham, Todd VanArsdale, David V. Shields, Nathan V. Lee, Maria Koehler, Kim Arndt. Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY34-03. doi:10.1158/1538-7445.AM2014-SY34-03

Abstract: The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, loss of function of PTEN or activation of receptor tyrosine kinases. Inhibition of key signaling proteins in the pathway, such as PI3K, AKT and mTOR, therefore represents a high value targeting strategy for diverse cancers. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in in vitro biochemical, cell and xenograft models. In in vitro biochemical assays PF-04691502 inhibited recombinant PI3Kα, β, γ and δ isoforms with Ki's of 1.2-2.2 nM and recombinant mTOR with a Ki of 9.1 nM. PF-04691502 demonstrated a high degree of selectivity for inhibition of PI3K family kinases as shown by lack of activity against a panel of & gt;75 protein kinases, including the Class III PI3K hVps34. PF-04691502 also inhibited transformation of avian fibroblasts mediated by PI3K γ, δ, mutant PI3Kα E545K or membrane-localized AKT with IC50's of ∼100nM. In cell assays PF-04691502 inhibited PI3K/mTOR signaling in SKOV3 ovarian cancer cells with PI3Kα mutations and in U87MG glioblastoma cells with PTEN alteration, as indicated by reduced levels of phosphorylation of AKT(T308), AKT(S473) and S6 ribosomal proteins. Functional studies for anti-proliferative effects suggest PF-04691502 has broad efficacy across tumor types. In SKOV3 and U87MG xenograft models PF-04691502 treatment resulted in dose-dependent tumor growth inhibition (TGI) with maximum TGI of ∼70% at the maximum tolerated dose of 10 mg/kg, by once daily oral dosing. Inhibition of AKT(S473) phosphorylation and S6RP(S235/236)/PRAS40(T246)/4EBP1(T37/46) phosphorylation were used as quantitative and qualitative pharmacodynamic (PD) endpoints, respectively; a clear pharmacokinetic (PK)/PD relationship was established in both models after multiple dose oral administration. In the U87MG xenograft model AKT(S473) phosphorylation was inhibited with an estimated EC50 of 5.7 nM (free plasma concentration) based on PK/PD modeling. The free plasma Area Under Curve was estimated to be 850 nM*hr for 70% TGI at 10mg/kg and was found to be similar in the SKOV3 model. The projected human efficacious dose of 10 mg once daily oral dosing provides Caverage steady state exposure of 22.4 nM (free plasma concentration) which is sufficient for 50-80% inhibition of pAKT S473, and corresponds to 74% TGI. Phase 1 clinical trials of PF-04691502 as a single agent are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4479.

Abstract: Nomination of new oncology targets has been greatly aided by advances in genetic screening and profiling, but developing potent, selective small molecule inhibitors against these targets remains a resource intensive pursuit. To significantly de-risk this process we applied a chemical biology strategy to model pharmacological inhibition of the MASTL kinase and showed that selective enzymatic inhibition accurately mimicked targeted genetic perturbation. Specifically, we engineered an electrophile-sensitive version of MASTL through a single amino acid substitution, Asp117Cys, within the ATP-binding site hinge region. Only 11 human kinases contain a cysteine handle in this particular hinge position (H10), which drastically limits the scope of off-targets prone to any H10 Cys-selective covalent inhibitors but also demonstrates that an H10 Cys is not incompatible with kinase activity. Furthermore, endogenous H10 Cys kinases include several targets of successful covalent inhibitor development campaigns (e.g. EGFR, JAK3 and BTK). This broad availability of optimized compounds allowed us to quickly screen and identify potent inhibitors of the MASTL Asp117Cys recombinant mutant. MASTL overexpression is observed across a broad spectrum of solid tumors, so to directly examine how its inhibition would impact cancer cell growth, Asp117Cys was created in the endogenous MASTL gene through CRISPR-directed gene editing. Pancreatic cancer cells homozygous for MASTL Asp117Cys showed clear dose-dependent growth inhibition when treated with a T790M mutant-specific EGFR covalent inhibitor (PF-06459988). Moreover, inhibitor treatment induced a visible phenotype of large multi-nucleated cells that phenocopies genetic perturbation of MASTL and aligns with MASTL's functional role regulating mitotic division. These inhibitor induced effects were not observed in unmodified parental cells and affinity purification experiments utilizing an alkyne probe of PF-06459988 confirmed binding specifically in engineered Asp117Cys mutant cells. When these Asp117Cys mutant cells are propagated as mouse xenografts, their in vivo tumor growth rate is indistinguishable from parental, but PF-06459988 treatment selectively induces tumor growth inhibition of mutant cells, thus demonstrating that selective enzymatic inhibition is sufficient to drive anti-tumor effects. Herein our application of an electrophile-sensitive mutant demonstrates how existing, optimized covalent small molecule inhibitors can be repurposed as chemical probes against engineered kinase domains. This engineered system provides a valuable orthogonal benchmark in advance of a drug discovery campaign, reveals target-dependent biology, and is likely to translate to additional kinase targets due to structural conservation within this enzymatic domain. Citation Format: Jon A. Oyer, Ted W. Johnson, Andrew C. Wang, Michael F. Maestre, Ana Flores-Bojorquez, Roksolana Melnychuk, Sergei Timofeevski, Sherry Niessen, Zhenxiong Wang, Jian Li, Wade C. Diehl, Koleen J. Eisele, Nathan V. Lee, Aihua Zou, Carl Davis, Eric C. Greenwald, Jacob DeForest, Martha Ornelas, Bryan Li, Stephanie Scales, Penney L. Khamphavong, Catherine M. Ambler, Yun Huang, Romelia Salomon-Ferrer, Samantha E. Greasley, Ben Bolanos, Neil Grodsky, Lawrence Lum, Todd L. VanArsdale, Indrawan J. McAlpine. Engineering electrophile-sensitive kinase mutants to accelerate oncology target validation [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 330.

Abstract: Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273–81. ©2016 AACR.

Abstract: PF-03084014, a small molecule γ-secretase inhibitor (GSI), has previously been shown to inhibit Notch signaling and demonstrated antitumor efficacy. The aim of this report is to gain insights into the mechanism PF-03084014-induced activity. The cell-based evaluation of PF-03084014 across 30 breast cancer (BC) cell lines indicated significant growth inhibition in only a subset of cell lines (3/30). In contrast, PF-03084014 demonstrated cell cycle arrest and induction of apoptosis by FACS analysis across the majority of a panel of T-acute lymphoblastic leukemia (T-ALL) cell lines. In vivo BrdU uptake of tumor and [18F]FLT-PET imaging studies demonstrated that PF-03084014 at an efficacious dose (120 mg/kg) caused cell cycle arrest in Sup T1 tumors but not in MDA-MB-231 xenografts, although PF-03084014 suppressed the expression of Notch target genes and tumor growth in both models. These results suggest that the primary mechanism(s) for PF-03084014-induced efficacy in T-ALL and BC models may be different, possibly because the tumor-host microenvironment in the BC model plays an important role during Notch signaling activation. IHC analysis in a panel of solid tumors demonstrated that Notch ligands including jagged 1, 2 and Dll4 were predominately expressed in the host vasculature and stromal cells. To investigate the potential antiangiogenic activity of PF-03084014, an in vitro endothelial cell/fibroblast co-culture tube formation assay was utilized. PF-03084014 disrupted the multicellular lumen-like structures at 100 nM, indicating defective differentiation of endothelial cells in early stage angiogenesis. Ultrasound imaging was utilized to further investigate the antiangiogenic properties of PF-03084014 including both the quantitative measurement of the total tumor vessel volume using 3-D scanning methods and an assessment tumor blood vessel function by measuring blood flow after iv injection of microbubble contrast agents. When MDA-MB-231 tumor bearing mice were administered with an efficacious dose of PF-03084014 consecutively for 4 days, ultrasound imaging revealed a significant (p & lt; 0.05) decrease of total tumor vessel volume and function compared to the vehicle treatment. A subsequent FITC-lectin vascular perfusion assay was performed to confirm the reduction in functional microvasculature by PF-03084014. In addition to the antiangiogenic effect, we also observed treatment-induced caspase 3 activation and Ki67 suppression in the MDA-MB-231 tumor model (2 days after treatment), suggest the GSI also induces apoptosis and cytoreductive activity. Collectively, our data provide insights that activation of Notch signaling requires the interaction of tumor cells with their host environment in solid tumors, and impairing Notch signaling by PF-03084014 results in significant antitumor efficacy in the breast cancer xenografts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5218.