Yimin Qian

PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer

Significance We describe the development of a small molecule that mediates the degradation of bromodomain and extra-terminal (BET) proteins and its application in the treatment of castration-resistant prostate cancer (CRPC). Few therapeutic options exist to treat CRPC, especially CRPC tumors expressing constitutively active androgen receptor (AR) splice variants that lack the ligand-binding domain and can effect androgen-independent transactivation of target genes. Importantly, we demonstrate that targeted degradation of BET proteins using proteolysis-targeting chimera (PROTAC) technology causes cell death in cultured prostate cancer cells and results in tumor growth inhibition or regression in mouse models of CRPC, including models that express high levels of AR splice variant 7. Our work thus contains a significant potential therapeutic advance in the treatment of this cancer. Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry ‘CyTOF’ analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.

The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.

BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Protein targeting chimeric molecules specific for bromodomain and extra-terminal motif family proteins are active against pre-clinical models of multiple myeloma

Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.

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

Abstract S4-03: Targeted and selective degradation of estrogen receptor (ER) alpha by PROTACs

ERα-positive breast cancers comprise approximately 80% of all newly diagnosed cases. Current treatment approaches targeting ER signaling include antagonizing and/or downregulating ER or reducing estrogen levels. Faslodex (fulvestrant) is the only clinically-approved agent that is both a potent ER antagonist and downregulator but has limitations given its pharmacokinetics and route of administration. Over the past several years, targeted ER therapies have focused on developing selective estrogen receptor downregulators (SERDs, i.e, GDC-0810, GDC-0927, AZD9496, RAD1901). The mechanisms involved in ER downregulation by SERD binding are not completely understood, but evidence suggests that conformational changes in the receptor upon ligand binding combined with specific co-regulator interactions destabilize the receptor making it a target for passive proteasomal degradation. We hypothesized that the complex ER pharmacology required for SERD-based passive degradation might be different across various ER-positive cell lines and that targeted degradation of the receptor by actively recruiting the ubiquitin-proteasome machinery would provide a better approach for reducing ER levels. To test this hypothesis, we developed potent molecules directed against ER using our pioneering technology—proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional molecules that actively recruit specific E3 ligases resulting in ubiquitylation and degradation of target proteins. When testing for ER degradation using several SERDs and ER PROTACs, we discovered that both fulvestrant and ER PROTACs provided robust degradation in all ER-positive lines ( 90% reduction) whereas other SERDs did not degrade or only modestly degraded the receptor. Importantly, MCF-7 cells were uniquely sensitive to SERD-based degradation of ER compared to other cell lines. Subcutaneous administration of fulvestrant (1mpk) or ER PROTACs (10 mpk) reduced uterine ER alpha levels in immature rats (>65% reduction). PROTAC-mediated degradation of ER was also achieved in breast cancer xenografts. To further validate the PROTAC mechanism, incubation of ER-positive cells with ER PROTACs resulted in increased levels of poly-ubiquitylated ERα when compared to SERDs. Lastly, to demonstrate the specificity of PROTAC-mediated ERα degradation, we utilized a cellular expression proteomics-based approach to examine over 7,000 proteins. In this experiment, only ERα and several known proteins whose genes are regulated by ERα, were significantly reduced by PROTACs. It remains to be seen how the current class of investigational downregulators will perform in the clinic. More importantly, a better understanding of the therapeutic potential and benefit of degrading the receptor instead of inhibiting the receptor needs to be explored. To that end, we continue to develop and characterize novel ER PROTACs with the anticipation that targeted ERα degradation will provide a greater clinical benefit than receptor antagonism. Citation Format: Flanagan JJ, Rossi AK, Anderoli M, Willard RR, Gordon D, Harling J, Churcher I, Smith I, Zinn N, Bantscheff M, Crews CM, Crew A, Coleman KG, Winkler JD, Qian Y. Targeted and selective degradation of estrogen receptor (ER) alpha by PROTACs [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S4-03.

Abstract 4699: Superior lethal activity of single agent BET protein PROTAC compared to bromodomain inhibitor or in combination with JAK inhibitor against post-myelofibrosis secondary AML cells

Myeloproliferative neoplasm, myelofibrosis (MPN-MF), exhibits increased JAK-STAT signaling and often progresses (∼15-20%) to AML (sAML). JAK inhibitor (JAK-I) ruxolitinib (Rux) or standard induction chemotherapy is only modestly active against sAML, culminating in treatment-refractory relapse. Genetic alterations commonly documented in sAML include the co-occurrence of JAK2 V617F and mutant TP53. Here, we demonstrate that treatment with ARV-825 (Arvinas, Inc.), a BET (bromodomain and extraterminal) protein PROTAC (proteasome activating chimera) which degrades (through the proteasome) BET proteins by binding and recruiting E3 Ubiquitin ligase cereblon activity to them, caused efficient, and prolonged depletion of the levels of the BET protein BRD4. In contrast, treatment with the bromodomain inhibitors (BET-Is) JQ1 or OTX015 induced the levels of BRD4. ARV-825 treatment also mediated greater and more sustained attenuation than OTX-015 of the mRNA and protein expressions of BCL-xL, CDK4/6, PIM1, p-STAT5 and p-STAT3 levels, while concomitantly inducing p21 and p27 in the cultured sAML (HEL92.1.7 and SET2) cells. This correlated with high level of apoptosis in the cultured and patient-derived post-MPN-MF sAML cells, with relative sparing of the normal CD34+ progenitor cells. Compared to treatment with each agent alone, co-treatment with ARV-825 and the JAK-Is Rux (100 to 1000 nM) or pacritinib (250 to 1000 nM), was synergistically more lethal against the cultured sAML cells (CI of 10-fold resistant to ruxolitinib; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAK-I. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to both ARV-825 and AUY922. Furthermore, co-treatment with ARV-825 and AUY922 was also synergistically lethal against HEL/JIR cells (CI Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Jing Lu, Yimin Qian, Kanak Raina, Baohua Sun, Stephanie S. Krieger, Simrit Parmar, Srdan Verstovsek, Kapil N. Bhalla. Superior lethal activity of single agent BET protein PROTAC compared to bromodomain inhibitor or in combination with JAK inhibitor against post-myelofibrosis secondary AML cells. [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 4699.