Kevin Coleman

Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271

Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC(50) of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC(50) of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100x selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC(50) of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.

Identification of CDK4 Sequences Involved in Cyclin D1 and p16 Binding

Activation of CDK4 is regulated, in part, by its association with a D-type cyclin. Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16 INK4A . To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16 INK4A we performed site-directed mutagenesis of CDK4. The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus. In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus. All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.

PF-03814735, an Orally Bioavailable Small Molecule Aurora Kinase Inhibitor for Cancer Therapy

The Aurora family of highly related serine/threonine kinases plays a key role in the regulation of mitosis. Aurora1 and Aurora2 play important but distinct roles in the G2 and M phases of the cell cycle and are essential for proper chromosome segregation and cell division. Overexpression and amplification of Aurora2 have been reported in different tumor types, including breast, colon, pancreatic, ovarian, and gastric cancer. PF-03814735 is a novel, potent, orally bioavailable, reversible inhibitor of both Aurora1 and Aurora2 kinases that is currently in phase I clinical trials for the treatment of advanced solid tumors. In intact cells, the inhibitory activity of PF-03814735 on the Aurora1 and Aurora2 kinases reduces levels of phospho-Aurora1, phosphohistone H3, and phospho-Aurora2. PF-03814735 produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. Although PF-03814735 produces significant inhibition of several other protein kinases, the predominant biochemical effects in cellular assays are consistent with inhibition of Aurora kinases. Once-daily oral administration of PF-03814735 to mice bearing human xenograft tumors produces a reduction in phosphohistone H3 in tumors at doses that are tolerable and that result in significant inhibition of tumor growth. The combination of PF-03814735 and docetaxel in xenograft mouse tumor models shows additive tumor growth inhibition. These results support the clinical evaluation of PF-03814735 in cancer patients. Mol Cancer Ther; 9(4); 883–94. ©2010 AACR.

Discovery and Pharmacologic Characterization of CP-724,714, a Selective ErbB2 Tyrosine Kinase Inhibitor

Amplification and overexpression of erbB2 (Her-2/neu) proto-oncogene has been linked to human malignancies including tumors of the breast, ovary, and stomach. It has been implicated in tumor growth, sensitivity to standard chemotherapy, prognosis of patients, and disease-free survival. Although the clinical use of trastuzumab (Herceptin) has prolonged the survival of breast cancer patients with erbB2-overexpressing tumors, there is an urgent need for more potent and orally bioavailable small-molecule inhibitors. CP-724,714 is a potent inhibitor of erbB2 receptor autophosphorylation in intact cells and is currently undergoing phase I clinical trials. Here, we describe the effects of CP-724,714 in vitro and in vivo in human breast cancer models. CP-724,714 is selective for inhibiting growth of HER2-driven cell lines. In addition, we show that it induces G1 cell cycle block in erbB2-overexpressing BT-474 human breast carcinoma cells and inhibits erbB2 autophosphorylation in xenografts when administered p.o. to athymic mice. It induces a marked reduction of extracellular signal-regulated kinase and Akt phosphorylation, tumor cell apoptosis, and release of caspase-3. P.o. administration (q.d. or b.i.d.) of CP-724,714 inhibits the growth of erbB2-overexpressing tumors in athymic mice without overt adverse effects.

Regulation of Transforming Growth Factor α Expression in a Growth Factor-Independent Cell Line

ABSTRACT Aberrant transcriptional regulation of transforming growth factor α (TGFα) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGFα expression in the malignant phenotype. In this paper, we report on TGFα promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGFα and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGFα. However, constitutive expression of TGFα antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGFα mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGFα autocrine activation is the major stimulator of TGFα expression in this cell line, TGFα promoter activity should be reduced in the antisense TGFα clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGFα promoter was restored in an antisense-TGFα-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system, we were able to identify a 25-bp element within the TGFα promoter which conferred TGFα autoregulation to the TGFα promoter in the HCT116 cell line. In the TGFα-antisense-RNA-expressing clones, this element was activated by exogenous EGF. This 25-bp sequence contained no consensus sequences of known transcription factors so that the TGFα or EGF regulatory element within this 25-bp sequence represents a unique element. Further characterization of this 25-bp DNA sequence by deletion analysis revealed that regulation of TGFα promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall expression of the activators is pivotal in determining the level of response to EGF or TGFα stimulation. The specific nuclear proteins binding to this region are also regulated in an autocrine-TGFα-dependent fashion and by exogenous EGF in EGF-deprived TGFα antisense clone 33. This regulation is identical to that seen in the growth factor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation oftrans-acting factor binding by EGF suggests that the effect is directly due to growth factor and not mediated by changes in growth state. We conclude that this element appears to represent the major positive regulator of TGFα expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of TGFα promoter activity in the growth factor-independent phenotype.

Aberrant Regulation of Transforming Growth Factor-α during the Establishment of Growth Arrest and Quiescence of Growth Factor Independent Cells

Autocrine transforming growth factor α (TGFα) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFα confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFα mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFα mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at −201 to −225 within the TGFα promoter which mediates the differential regulation of TGFα expression during quiescence establishment in these two cell lines. This same sequence confers TGFα promoter responsiveness to exogenous growth factor or autocrine TGFα. The abberant upregulation of TGFα mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFα approaches showed that the dysregulated TGFα expression in quiescent HCT116 cells is a function of the strong TGFα autocrine loop (not inhibited by blocking antibodies) in these cells.

Abstract PR08: ARV-330: An androgen receptor PROTAC degrader for prostate cancer

Patients with prostate cancer who progress on therapy often have enhanced Androgen Receptor (AR) signaling due to several mechanisms: increased androgen production, increased AR expression, and/or specific AR mutations that render current therapies ineffective. A novel approach to block AR signaling is to specifically target AR for degradation. To do this, we have created AR PROTACs (PROtein-TArgeting Chimeras), bi-functional molecules that have an AR binding moiety on one end and an E3 ligase-recruiting element on the other end, which leads to AR ubiquitination and degradation. We have applied this technology to determine whether it could address mechanisms of resistance to current therapy in prostate cancer models. Our lead AR PROTAC, ARV-330, degrades AR in LNCaP and VCaP cells with 50% degradation concentrations (DC50s) 80% after sc injection. Treatment of mice with ARV-330, at doses ranging from 0.3 to 10 mg/kg, resulted in reduction of AR protein levels and prostate involution in normal mice and, in mice implanted with VCaP tumors, reduction in plasma PSA and blockade of tumor growth. In summary, the AR PROTAC ARV-330 removes AR from prostate cancer cells in a potent manner and produces therapeutic effects as a result. This cellular efficacy has translated into biomarker activity and efficacy in animal models, and ARV-330 is now in preclinical development. Thus, targeted degradation of AR may provide a novel mechanism for providing efficacious therapy for patients with prostate cancer for whom current therapies have failed. Citation Format: James D. Winkler, Meizhong Jin, Andy P. Crew, AnnMarie K. Rossi, Ryan R. Willard, Hanqing Dong, Kam Siu, Jing Wang, Deborah A. Gordon, Xin Chen, Caterina Ferraro, Craig M. Crews, Kevin Coleman, Taavi K. Neklesa. ARV-330: An androgen receptor PROTAC degrader for prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PR08.

Abstract LB-097: Targeted degradation of the androgen receptor in prostate cancer

Progression of prostate cancer in patients treated with anti-androgen therapy usually involves several mechanisms of enhanced Androgen Receptor (AR) signaling, including increased intratumoral androgen synthesis, increased AR expression and AR mutations. We have developed a protein degradation technology called PROTACs (PROteolysis TArgeting Chimera), which uses bi-functional molecules that simultaneously bind a target of choice and an E3 ligase. PROTACs, via induced proximity, cause ubiquitination and degradation of the targeted, pathological protein. As opposed to traditional target inhibition, which is a competitive process, degradation is a progressive process. As such, it is less susceptible to increases in endogenous ligand, target expression, or mutations in the target. Thus this technology seems ideal for addressing the mechanisms of AR resistance in patients with prostate cancer. AR PROTACs were shown to degrade AR in LNCaP and VCaP cells, with low nM to pM potency, and had a >85% reduction in AR concentration (Dmax). Degradation was rapid, with 50% of AR lost within 15 minutes and maximal degradation observed by 4 hours. The degradation process in cells was specific, as the PROTAC activity can be competed with excess E3 ligand and PROTACs with an inactive epimer for E3 ligase binding did not degrade AR. AR PROTACs induced rapid apoptosis and cell death in VCaP cells. In LNCap and VCaP cell systems, AR PROTACs were anti-proliferative under conditions in which enzalutamide was inactive, such as increasing concentrations of the AR agonist R1881 and cells containing the ARF876L mutation. AR PROTACs typically exhibited good pharmacokinetic properties, with t1/2 values of several hours and bioavailability of >50% after ip or sc injection. In mice, AR PROTACs demonstrate in vivo activity, including reduction of AR protein levels, prostate involution and tumor growth inhibition. In summary, PROTACs designed to degrade AR are potent, specific, active in vitro and in vivo, and have cellular efficacy superior to enzalutamide. Targeted degradation of AR may provide a novel mechanism for providing efficacious therapy for patients with prostate cancer for whom current therapies have failed. Citation Format: Meizhong Jin, James D. Winkler, Kevin Coleman, Andrew P. Crew, AnnMarie K. Rossi, Ryan R. Willard, Hanqing Dong, Kam Siu, Jing Wang, Deborah A. Gordon, Xin Chen, Caterina Ferraro, Craig M. Crews, Taavi K. Neklesa. Targeted degradation of the androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-097. doi:10.1158/1538-7445.AM2015-LB-097

Abstract 5076: IHC and flow cytometry quantifies BRD4 levels in surrogate tissues after ex-vivo and in-vivo dosing with a BRD4 degrading PROTAC

The Bromodomain and Extra-Terminal (BET) protein BRD4 has been identified as a key transcriptional regulator of various oncogenes, most notably MYC, in numerous human malignancies. Efforts have been extensive over recent years to inhibit the gene regulatory action of BRD4 and BET inhibitors have entered clinical trials. In a new approach to cancer therapeutics, we have developed a proteolysis-targeting chimera (PROTAC) molecule, ARCC-29, which induces degradation of BET (BRD4/3/2) proteins via the proteasome. ARCC-29 degrades BRD4 in several human malignant cell lines and in in-vivo rodent xenograft tumors. In mice xenograft models of prostate (22Rv1), DLBCL (SU-DHL-6) and ovarian (A2780) tumors, this molecule demonstrates a strong PK/PD/efficacy relationship. Obtaining tumor tissue from patients often requires invasive procedures and may be unavailable. We have developed two assays using surrogate tissues to evaluate PROTAC mechanism of action which could be applied in the clinical setting. We have developed a flow cytometry protocol to quantitatively assess intra-cellular BRD4 levels in peripheral blood mononuclear cells (PBMCs). Frozen or fresh human PBMCs were cultured for six hours ex-vivo with ARCC-29, fixed and permeabilized for intra-cellular BRD4 staining, and BRD4 levels quantified in gated lymphocytes. ARCC-29 reduced BRD4 levels to 13, 10, 16, and 9 percent of vehicle in four fresh PBMC samples, and to 10, 9, 11, 6, 8 and 35 percent in six frozen PBMC samples. Treatment of PBMCs with the BRD4 inhibitor OTX-015, showed similar or increased levels of BRD4 compared to vehicle control. Inactive stereoisomers of ARCC-29 also showed BRD4 levels increased or similar to vehicle control. Duplicates of selected experiments were analyzed by western blot, corroborating the depletion of BRD4. The second assay uses immunohistochemistry (IHC) to evaluate BRD4 degradation in skin biopsies of Sprague Dawley and nude male rats dosed with 2mg/kg intravenous (i.v.) BRD4 PROTAC ARCC-29. IHC analysis of rat skin eight hours after in-vivo dosing with ARCC-29 demonstrated a dose-dependent reduction in BRD4 protein. Rats dosed once a week with ARCC-29 [2 mg/kg] showed a 60-90% reduction in BRD4 levels compared to vehicle, while 0.3 mg/kg twice per week showed a 10-30% decrease in BRD4 levels. These experiments demonstrate that BRD4 degradation can be evaluated in surrogate tissue. Flow cytometry using PBMCs and skin IHC provide potential alternative methods for confirming the PROTAC mechanism-of-action in the clinic using pre- and post-treatment patient samples. DISCLOSURES All authors are employed by Arvinas, LLC. Citation Format: Sheryl M. Gough, Kanak Raina, Debbie Gordon, Ryan Willard, Martha Altieri, Angela Shen, Yimin Qian, Taavi Neklesa, Kevin Coleman, Ian Taylor. IHC and flow cytometry quantifies BRD4 levels in surrogate tissues after ex-vivo and in-vivo dosing with a BRD4 degrading PROTAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5076. doi:10.1158/1538-7445.AM2017-5076

Abstract 5067: BET protein proteolysis targeting chimera (BETP-PROTACs) exert more potent activity than BETP bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells

In BCR-ABL1-negative myeloproliferative neoplasms with myelofibrosis (MPN-MF) transformation to AML (sAML) occurs in up to 20% of patients. Ruxolitinib (R) is a type I, ATP-competitive, JAK1 & 2 inhibitor (JAKi), which is effective in the therapy of MPN-MF but does not significantly impact the clinical outcome in post-MPN sAML. We have previously reported that treatment with BETi, e.g. JQ1 or OTX015 inhibits growth and induces apoptosis of cultured sAML cells, including those that express JAK2 V617F and mutant TP53, e.g. HEL92.1.7 and SET2, as well as patient-derived (PD) CD34+ sAML cells. BETi treatment attenuated the protein expressions of c-MYC, p-STAT5, Bcl-xL, CDK4/6, PIM1 and IL-7R, while concomitantly inducing the levels of HEXIM1, p21, NOXA and BIM in the sAML cells. However, treatment with BETi leads to the accumulation of BETP, e.g. BRD4, which may reduce BETi-mediated repression of c-MYC, NFκB and BETP-regulated oncoproteins. In contrast, BETP-PROTACs (proteolysis targeting chimera) ARV-825 and ARV-771 (Arvinas Inc.) degrade BETPs (including BRD4) in the cultured and PD CD34+ sAML cells. At equimolar concentrations, BETP-PROTACs were significantly more potent than the BETi in inducing apoptosis of cultured and PD sAML cells (p 90%) in the sAML cells. BETP-PROTAC treatment caused more up and down regulation of mRNA and protein expressions than BETi, as determined by RNA-Seq and reversed phase protein array (RPPA) analyses, respectively. As compared to treatment with BETi, BETP-PROTAC caused greater depletion of c-MYC, JAK2, p-STAT5, STAT5, p-STAT3, STAT3, PIM1 and Bcl-xL, whereas the protein levels of p21 and p27 were upregulated. CyTOF or mass-cytometry also showed that BETP-PROTAC, more than OTX015 treatment, reduced BRD4, c-MYC and p-Rb, while inducing p21 levels in the CD34+ sAML stem/progenitor cells expressing CD90, CD244, CD123 and TIM3-Fc. Compared to treatment with each agent alone, co-treatment with BETP-PROTAC and R was synergistically lethal against the cultured and PD CD34+ sAML cells. Additionally, co-treatment with BETP-PROTAC and HSP90 inhibitor AUY922 or BCL2/BcL-xL antagonist ABT263 was synergistically lethal against R-sensitive and R-resistant sAML cells. As compared to treatment with vehicle control, R treatment alone, treatment with BETP-PROTAC ARV-771 alone or in combination with R significantly reduced the in vivo sAML burden and improved the median survival of the immune-depleted mice engrafted with luciferase-transduced HEL92.1.7 cells. These findings strongly support further in vivo development of the novel BETP-PROTACs-based combinations against post-MPN sAML. Note: This abstract was not presented at the meeting. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Kanak Raina, Taghi Manshouri, Kevin G. Coleman, Yimin Qian, Andrew P. Crew, Angela Shen, Christopher P. Mill, Baohua Sun, Misun Kim, Agnieszka J. Nowak, Srdan Verstovsek, Craig M. Crews, Kapil N. Bhalla. BET protein proteolysis targeting chimera (BETP-PROTACs) exert more potent activity than BETP bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5067. doi:10.1158/1538-7445.AM2017-5067