Targeting the C481S Ibrutinib-Resistance Mutation in Bruton's

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Targeting the C481S Ibrutinib-Resistance Mutation in Bruton’s Tyrosine Kinase using PROTAC-mediated Degradation Alexandru D. Buhimschi, Haley A Armstrong, Momar Toure, Saul Jaime-Figueroa, Timothy L Chen, Amy M. Lehman, Jennifer A Woyach, Amy J. Johnson, John C Byrd, and Craig M Crews Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00391 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Biochemistry

Targeting the C481S Ibrutinib-Resistance Mutation in Bruton’s Tyrosine Kinase using PROTAC-mediated Degradation Alexandru D. Buhimschi1,†; Haley A. Armstrong2,†; Momar Toure1; Saul JaimeFigueroa1; Timothy L. Chen3, Amy M. Lehman4; Jennifer A. Woyach2,3, Amy J. Johnson3, John C. Byrd2,3,‡, Craig M. Crews*,1,5,6,‡ 1

Department of Molecular, Cellular, and Developmental Biology, Yale University

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Division of Pharmaceutics & Pharmaceutical Chemistry, College of Pharmacy, The

Ohio State University 3

Department of Internal Medicine, Division of Hematology, The Ohio State University

4

Center for Biostatistics, The Ohio State University

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Department of Chemistry, Yale University

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Department of Pharmacology, Yale University

†

These authors contributed equally to this work

‡

These two senior authors contributed equally to this work

* Corresponding author Correspondence to: Craig M. Crews, PhD Department of Molecular, Cellular, and Developmental Biology Yale University Kline Biology Tower (KBT) 400 New Haven, CT, 06511 Telephone: (203) 432-9364 Fax: (203) 432-6161 Email: [email protected] or John C. Byrd, MD Division of Hematology, Department of Internal Medicine The Ohio State University Columbus, Ohio 43210 Email: [email protected] KEYWORDS BTK | PROTAC | Protein Degradation | CLL | C481S

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ABSTRACT Inhibition of Bruton’s tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Yet, more than 80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates based on its potency to induce BTK knockdown. MT802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib and retains equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL.


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SIGNIFICANCE We have developed a small molecule PROteolysis TArgeting Chimera (PROTAC) for the degradation of wild type and C481S mutant forms of Bruton’s tyrosine kinase (BTK). The C481S mutation is the most prevalent form of resistance to the irreversible BTK inhibitor ibrutinib when it is administered as first-line therapy for CLL. As of yet, no effective treatment options exist for patients with disease progression due to this mutation and their outcome is poor. We show that our PROTAC potently degrades both wild-type and C481S BTK, binds fewer off-target kinases than ibrutinib, and impairs BTK signaling in primary B-cells isolated from C481S patients. Further studies will be necessary to assess the in vivo applicability of our approach, but this is the first report on the potential advantages of protein degradation for addressing this form of CLL resistance. Our work, thus, applies PROTACs to a new disease class with potential to improve how resistant CLL could be managed and treated in the future.

INTRODUCTION Targeted cancer therapy directed at kinase inhibition has been successful for a multitude of diseases where mutated or fusion transcript proteins are present and overactive1. Recently, kinases relevant to pathway dependency in select cancers have been exploited. Specifically, B-cell receptor (BCR) signaling has been shown to be constitutively active in compartments of CLL proliferation (bone marrow, spleen, and lymph node) among all CLL patients, and enhanced even further among patients with the more aggressive form of the disease evidenced by ZAP-70 over-expression2, 3. A variety of kinases involved in proximal BCR signaling, including spleen tyrosine kinase (Syk), phosphatidylinositide 3-kinase-δ (PI3K-δ), and Bruton’s tyrosine kinase (BTK) are


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potentially targetable with small molecule inhibitors.

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Our group has focused most

heavily on BTK due to the availability of clearly delineated loss of function and kinasedead murine models. Mice where BTK has been inactivated show impaired B-cell maturation as their primary phenotype. This is in contrast to the other potentially promising CLL drug targets mentioned above: loss of Syk is embryonically lethal, and PI3K-δ-inactive mice have a broad range of immune phenotypes, including impaired thymocyte development4.

To illustrate the therapeutic promise of targeting these

kinases, crossing PI3K-δ or BTK-inactive mice with CLL mouse models prevented the disease, but survival was nonetheless impaired in the case of the former due to alternative on-target effects promoting both infections and colitis. The progeny of the BTK-inactive crossing were, on the other hand, phenotypically normal. Through such studies and others, BTK has emerged as an important drug target in CLL5. BTK is a Tec family kinase of hematopoietic origin found in B-cells throughout their development, where it propagates proximal B-cell receptor (BCR) signaling6. When the BCR is stimulated by antigen, Syk is first activated to induce BTK phosphorylation and activation7. BTK then drives multiple pro-survival and proliferative pathways, including the activation of PLCγ-2 to release intracellular calcium stores as well as the Ras/Raf/MEK/ERK kinase pathway8,9. In turn, factors such as NF-κB localize to the nucleus and induce transcription of growth factors and anti-apoptotic proteins that enhance survival and drive proliferation10. The predominant approach for targeting BTK has been via small-molecule mediated inhibition11-14. To date, the most successful clinical implementation of a direct BTK inhibitor has been that of ibrutinib, which irreversibly binds to cysteine 481 in the kinase domain of BTK15,16. Inhibition of BTK with ibrutinib is prolonged and has resulted


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in both dramatic and durable responses across virtually all patients treated. However, many patients receiving ibrutinib therapy experience disease relapse, which has been attributed mainly to a mutation in BTK that only allows for reversible binding of ibrutinib to the kinase: specifically, substitution of cysteine 481 with serine abolishes ibrutinib’s ability to covalently bind to BTK17,18. Because ibrutinib has a relatively short in vivo halflife, BTK function within the resistant tumor cell is partially restored, facilitating tumor growth and eventual clinical relapse. The outcomes of CLL patients developing C481S mutant CLL and clinical disease progression are poor19. That the ibrutinib-impairing C481S mutation in BTK is consistently observed in CLL patients suggests that these cell populations still require BTK signaling to survive and proliferate. Consistent with this expectation, C481S mutant CLL is still responsive to BTK-targeted therapy, indicating that an agent with retained efficacy in this mutational setting should control disease and continue to exploit the aforementioned benefits of BTK susceptibility in CLL20. Herein, we sought to apply a new therapeutic approach to the treatment of CLL, including those instances with the C481S mutation. Originally developed by our group in 2001, PROTACs are a class of chemical agents that target specific proteins for ubiquitination and degradation by the proteasome21-24. This is achieved by employing a pharmacological agent to recruit the targeted protein to a specific E3 ubiquitin ligase complex, which would not normally bind to the target protein, resulting in target protein ubiquitination. Conceptually, PROTACs are composed of three structural elements: 1) a ligand for an E3 ubiquitin ligase; 2) a ligand for the target protein to be degraded; and 3) a flexible linker joining the aforementioned two ligands. PROTACs were first successfully implemented using peptidic E3 ligase ligands, but these early PROTACs were limited due to poor cell


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permeability stemming from their large sizes25,26. Development of the technology was greatly accelerated when we reported a set of potent small-molecule PROTACs based on a newly designed and developed ligand for the von Hippel-Lindau (VHL) E3 ligase2729

. Since these pioneering reports, the set of proteins successfully targeted by

PROTACs continues to grow and includes: RIPK2 and ERRα, BRD4, BCR/Abl and Abl kinases, and recently several receptor tyrosine kinases30-36. The above targets have been degraded with several E3 ligases that have been shown to be amenable for PROTAC development, which also include cereblon (using thalidomide analogs) and cIAP (shown with SNIPERs, a subclass of PROTACs) in addition to VHL37-42. Traditional inhibitors work by an “occupancy-driven” paradigm, whereby efficacy is dictated by prolonged binding to a target protein at sites which abrogate the target’s necessary functions. PROTACs, on the other hand, work through “event-driven” pharmacology, whereby only transient binding that is sufficient to induce recruitment to an E3 ligase can result in a biological consequence (i.e. degradation of the target protein). The immediate advantages of this paradigm are two-fold: 1) binding may occur at any site on the target protein and 2) PROTACs can act catalytically to bind and degrade multiple target proteins enabling potential for lower drug dosages needed to observe pharmacological effect30. Recently, our group and others have shown that small molecule PROTACs can induce in vivo degradation in picomolar to nanomolar concentration ranges, effectively transitioning what was once considered a chemicalbiology concept into a therapeutically-promising platform for drug development30 43. Since BTK has been well-validated as a therapeutic target in CLL, we sought to extend the PROTAC platform to this critical signaling kinase and assess the merits of its degradation in comparison with inhibition. In particular, we reasoned that a PROTAC


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Biochemistry

should retain activity against C481S BTK due to its mechanism of action relying on a transient, reversible association with its substrate to induce ubiquitination and degradation. This study would serve to exemplify a disease context where the degradation mechanism of PROTACs is beneficial for evading mutations that cause relapse in response to inhibitors.

MATERIALS AND METHODS i. Culturing of Cancer Cell Lines and Patient Primary Cells NAMALWA and Jurkat cell lines were purchased from ATCC and cultured according to supplier guidelines at 37ºC, 5% CO2 in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum and 100 µg/mL streptomycin and 100 U/mL penicillin-G (Gibco). Wild-type and C481S BTK XLA cell lines were cultured as previously described44. Written, informed consent was obtained prior to the collection of cells from CLL patients using the IWCLL2008 criteria. Isolation of mononuclear cells from peripheral blood was conducted using density gradient centrifugation. B-cells were then negatively selected. Cells were then cultured at 1 x 107 cells/mL in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (VWR), 100 µg/mL streptomycin (Gibco), 100 U/mL penicillin-G (Gibco), and 2 mmol/L L-glutamine (Gibco). Cells were maintained at 37°C in 5% CO2.

ii. Cell Treatment and Immunoblotting For immortalized cell lines, 2 x 106 cells per PROTAC treatment condition were collected and washed once with ice cold PBS, followed by lysis in buffer containing 20 mM Tris (pH 8.0), 0.25% sodium deoxycholate, 1% Triton X-100, supplemented with protease inhibitors (Roche) and phosphatase inhibitors (10 mM NaF, 2 mM Na3VO4, 10


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mM β-glycerophosphate, 10 mM Na-pyrophosphate). Lysates were centrifuged at 15,000 x g for 10 min at 4°C and supernatant was quantified for total protein content using the Pierce BCA Protein Assay (Thermo Fisher Scientific). 30 µg of protein were loaded onto SDS-PAGE gels (Bio-Rad), transferred onto nitrocellulose membranes, and probed with the specified primary antibodies overnight with rocking at 4°C in 1X TBS-T (TBS-Tween) containing 5% non-fat milk. HRP-conjugated secondary antibodies (Pierce) were incubated with the membranes for 1h at room temperature at 1:10,000 dilutions in 5% non-fat milk in 1X TBS-T. Imaging was performed using the ECL Prime chemiluminescent western blot detection reagents (GE Healthcare) followed by visualization with the Bio-Rad ChemiDoc imaging instrument. All western blots were subsequently processed and quantified using the accompanying Bio-Rad Image Lab software. Primary antibodies used were: anti-actin antibody (cat. #MA1-744) purchased from Thermo Fisher Scientific; anti-BTK (cat. #8547), anti-pBTK (Y223, cat. #5082), anti-ITK (cat. #2380), anti-GAPDH (cat. #5179), anti-IKZF1 (cat. #9034), and anti-IKZF3 (cat. #15103) antibodies purchased from Cell Signaling Technology. All antibodies were used at 1:1,000 dilutions in 5% non-fat milk in 1X TBS-T unless otherwise noted in supplier specifications. Patient primary cells studied in dose response experiments were treated at densities of 1 x 107 cells plated for 24 hours per condition. The baseline and relapsed patient samples were collected from ACD cryovials, thawed, and treated with 1 µM MT802 24 hours before lysis, and 1 µM ibrutinib 2 hours before lysis (followed by a 1 hour media washout to simulate in vivo drug metabolism). All primary patient samples were stimulated with anti-IgM (Jackson ImmunoResearch) 15 minutes prior to lysis. Primary cell lysates were prepared as previously described45. The cell suspension was kept on


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ice and agitated every 10 minutes for 30 minutes, followed by centrifugation for 10 minutes at 4°C. Protein quantification was performed for each supernatant using the BCA method (Thermo Fisher Scientific). 50 µg of each sample were loaded onto SDSpolyacrylamide gels and electrophoresed. Transfer of the proteins and blocking of membranes were performed as previously described45. Proteins were detected using the antibodies described above: anti-phospho-BTK (Abcam, cat. #ab68217), anti-BTK (Cell Signaling Technologies, cat. #8547), antiGAPDH (Cell Signaling Technologies, cat. #5179).

Antibodies used were diluted

1:1,000 in Blotto blocker (Thermo Fisher Scientific) and kept at 4ºC with constant agitation for 12 to 72 hours. The blots were washed with 1X TBS-T three times for 10 minutes with constant agitation, then incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnologies) diluted 1:5,000 in 5% non-fat milk in 1X TBS-T for 2 hours at 4°C with constant agitation. Prior to developing, the blots were again washed with 1X TBS-T three times for 10 minutes with constant agitation. Blots were developed using one of two chemiluminescent reagents: WesternBright (Advansta) or SuperSignal (Thermo Fisher Scientific). Quantification was performed using computer densitometry (AlphaView software).

iii. Chemical Reagents and Synthesis of PROTACs Ibrutinib for cellular and in vitro kinase inhibition assays was purchased as a 10 mM stock solution in DMSO from Selleckchem. Full synthesis method and chemical characterization of all compounds reported is provided in the chemical supplement.

iv. KINOMEscanTM Profiling and In vitro Kinase Competitive Binding Assays


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Ibrutinib and MT-802 were submitted as 1 mM stock solutions in neat DMSO to DiscoverX for the scanMax service, which screens compounds for binding against a panel of 468 kinases. Both compounds were screened at an assay concentration of 1.0 µM. The assay principle and design have been previously reported46. For KdELECTTM experiments, DiscoverX utilizes the same platform as that employed for the scanMax service, only expanded across 11 concentration points in duplicate. For KdELECTTM experiments, the highest concentration of compound employed was 3.0 µM. In vitro competition assays to measure binding to wild-type and C481S BTK were performed by Reaction Biology Corporation. IC50 values were determined by fitting a 10point dose response curve generated from successive 3-fold dilutions starting at either 10 µM (MT-802) or 1 µM (Ibrutinib and compound 1). This assay measures the ability for kinase to directly phosphorylate substrate in the presence of compound47. All competitive binding curves were generated in the presence of 10 µM ATP.

RESULTS i. Design and Discovery of Potent BTK PROTACs The first consideration for design of BTK PROTACs was identification of a suitable warhead to bind BTK. We focused on an ibrutinib-based scaffold, primarily due to the available structural characterization of its binding mode to BTK. Ibrutinib binds BTK covalently at cysteine 481 in the ATP binding pocket of the kinase48. Since a notable advantage of PROTACs over inhibitors is their catalytic mechanism, we did not incorporate the electrophilic acrylamide moiety of ibrutinib into our BTK PROTACs. Instead, we elected to build the linker from the solvent-exposed para- position of the piperidine ring in our reversible ibrutinib derivative (compound 1) (Figure S1A & B). As


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the E3 ligase-recruiting element for incorporation at the distal end of the linker, we selected pomalidomide, which binds to the E3 ubiquitin ligase cereblon (Figure S1A)38. The linker was attached to pomalidomide initially at the 4-carbon position on the phthalimide ring, consistent with previous reports incorporating this ligand into PROTACs. Our first-generation BTK PROTACs, MT-540 and MT-541 were designed with 12-atom linkers and showed nearly complete degradation of BTK at 1.0 µM (Table S1) in NAMALWA cells, a Burkitt’s lymphoma-derived B-lymphocyte cell line. Shortening the linker by a single atom (MT-781) increased potency, based on the concentration of compound needed to degrade 50% (the DC50) of the total pool of BTK. However, shortening the linker length even further to 8- or 5-atoms (MT-797 and MT-783, respectively) resulted in an inability for these compounds to degrade BTK. These observations are consistent with the expected ternary complex model that explains PROTAC action36,49. In this model, we expect that short linkers are insufficient to permit PROTAC binding to both BTK and cereblon simultaneously, such that there can be no induced complex formation necessary for ubiquitination. Given this model, we explored a novel linker position on the pomalidomide scaffold, which we proposed could facilitate a ternary complex with a more favorable configuration for degradation. This was confirmed when placement of the same 8-atom linker at the 5-position on the phthalimide ring of pomalidomide resulted in our most potent BTK PROTAC, MT-802 (Figure 1A). Interestingly, a relative increase in potency was also observed when the 12-atom linker was placed at the 5-position (e.g. MT-809 vs. MT-541), indicating that this vector may be generally more favorable for inducing a productive cereblon-BTK ternary complex. Docking of MT-802 into the crystal structures of BTK and cereblon


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showed that the 8-atom linker is nearing the minimal length needed to bridge the two binding sites (Figure S1C) and shorter linkers would be unable to bridge the gap without inducing steric constraints, which is consistent with our experimental observations (Figure S1D). Lastly, it is interesting to note that while we screened several other PROTACs synthesized based on our VHL ligand, we were unable to identify any degraders as effective as those within the pomalidomide-based series: VHL-utilizing BTK PROTACs caused only modest target degradation (DMax = 50%), and even that required treatment at 1.0-2.5 µM27,28. We speculate that the inability for VHL to ubiquitinate BTK as effectively as cereblon may be due to suboptimal ternary complex geometry and/or lysine accessibility, but this remains to be demonstrated. Further work is ongoing to understand BTK’s seemingly exclusive compatibility with cereblon and not VHL.


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Biochemistry


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Figure 1. (A) Chemical structures of ibrutinib, active PROTAC MT-802, and inactive control compound, SJF-6625. (B) BTK levels in response to dose escalations of MT802, SJF-6625, and ibrutinib in NAMALWA cell line after 24h treatment. (C) Time course of BTK degradation with 250 nM MT-802 in NAMALWA cells. Each time point


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was matched with a DMSO (vehicle) treated condition. (D) NAMALWA cells were pretreated with DMSO, epoxomicin (1 µM), MLN-4924 (1 µM), ibrutinib (25 µM), and pomalidomide (25 µM) for 2.5h before treatment with DMSO (vehicle) or 250 nM MT802 for 4h.

ii. MT-802 is a Potent and Rapid Degrader of BTK In our initial characterization experiments, we showed that MT-802 degrades BTK with a DC50 of 9.1 nM with maximal degradation being observed by 250 nM. Since PROTACs work via a ternary complex driven mechanism, a common observation for many PROTACs

is

the

“hook-effect”,

whereby

binary

species

(BTK:PROTAC and

PROTAC:cereblon) can predominate over the productive ternary complex at sufficiently high PROTAC concentrations, thereby resulting in reduced degradation. However, we did not observe any significant rebound in BTK levels (i.e. a “hook”) in cells treated with up to 2.5 µM MT-802 (Figure S2). PROTACs inducing ternary complexes with significant positive cooperativity would be expected to have an expansion in the concentration range of their maximal effect due to diminished presence of the unproductive binary complexes29,36,49,50. The lack of an observable hook-effect suggests that MT-802 induces a high affinity ternary complex with significant positive cooperativity. We next synthesized SJF-6625, an inactive version of MT-802 that is incapable of binding to cereblon due to methylation of the glutarimide ring of the pomalidomide moiety (Figure 1A). As expected, neither ibrutinib nor SJF-6625 were able to induce degradation of BTK (Figure 1B), demonstrating that binding to cereblon is required for MT-802’s mechanism of action.


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Since MT-802 could elicit complete BTK knockdown at 250 nM, we decided to employ this concentration of compound in our follow-up characterization experiments. At this concentration, we showed that MT-802 fully degrades BTK as early as 4 hours, with half of the total BTK degraded after approximately 50 minutes (Figure 1C & S3A). Pre-treatment with epoxomicin, a proteasome inhibitor, followed by treatment with MT802 did not result in BTK degradation, indicating that proteasome function is required for BTK knockdown51. The same was observed after pre-treatment with MLN-4924, an inhibitor of the NEDD8-activating enzyme which neddylates and thereby activates many cullin-RING ligases, including the cullin-4 based cereblon complex. The necessity for direct binding to both BTK and cereblon was shown by pre-treating cells with excess of either ibrutinib or pomalidomide, both of which rescued BTK levels in response to MT802 (Figure 1D & S3B). These assays demonstrate that MT-802 directly engages BTK and cereblon to engender knockdown in a proteasome-dependent manner. iii. Enhanced Kinase Selectivity by MT-802 over Ibrutinib Having demonstrated that MT-802 is capable of potent BTK degradation and established the bona fides of its mechanism, we wanted to assess the specificity of MT802’s binding within the kinome. In general, our group and others have shown that the potency of in vitro kinase binding decreases when the linker and E3-targeting moiety are appended to the parent warhead34,36. It is known that ibrutinib shows off-target inhibition of other kinases, particularly those with cysteines homologous to C481 in BTK52,53. Since MT-802 lacks the acrylamide moiety that binds C481, we reasoned that our PROTAC may bind fewer off-target kinases than does ibrutinib. If confirmed, this finding would be relevant to efforts to develop more specific BTK-targeting agents that are free of the negative side-effects of ibrutinib, which include adverse cardiac,


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gastrointestinal, and skin events54,55. To address this, we utilized KINOMEscanTM, the high-throughput, competition-based binding assay service provided by DiscoverX46. This assay reports binding as a “percentage of control”, where lower values represent higher levels of kinase binding. Using this assay, we screened ibrutinib and MT-802 in parallel at 1.0 µM against a panel of 468 human kinases (Figure S4A & B). Previously assembled datasets on ibrutinib’s kinome-wide inhibition showed reasonable correlation with our own dataset (Figure S4C). As expected, BTK was among the most maximally bound kinases by both compounds (0.0 and 0.25 % of control for ibrutinib and MT-802, respectively). The only other kinase in the Tec family that was thoroughly bound by both ibrutinib and MT-802 was TEC (1.9 and 3.6% of control, respectively) (Figure 2A). BMX showed weaker binding to both ibrutinib and MT-802, and while TXK could be highly bound by ibrutinib, MT-802 showed weaker engagement (3.8 and 63% of control, respectively) (Table S2). Of note, MT-802 also bound equally well to ERBB3 (0.0 % of control for both MT-802 and ibrutinib) in the KINOMEscanTM dataset, but this binding did not lead to ERBB3 degradation when tested in OVCAR8 cells (Figure S5). This example underscores our previous observation that effective target engagement does not always correlate with target degradation, and that other factors such as ternary complex affinity and lysine accessibility may also be relevant36. To identify those kinases where a differential for binding exists between MT-802 and ibrutinib, we performed a Bland-Altman difference analysis on the two KINOMEscanTM data sets (Figure 2B). Using this approach, we identified several proteins in the TK and STE kinase families that were significantly bound by ibrutinib (% of control < 10%) but poorly bound by MT-802 (% of control > 80%). The three kinases for which the greatest differential was observed were ITK, MKK7, and JAK3, all of which


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are known to be kinases inhibited by ibrutinib56. Since we performed our KINOMEscanTM at only a single concentration of 1.0 µM, we wanted to validate the binding data observed for the set of differentially inhibited kinases using full dose response curves. To do this, we applied the DiscoverX KdELECTTM platform to several of the kinases which showed a statistically significant difference in the level of binding between ibrutinib and MT-802 (Figure S6 & Table S3). For ITK, MKK7, and JAK3, ibrutinib gave Kd values in the low nanomolar range, while MT-802 showed no ability to bind these kinases in the range of concentrations employed in the dose response (Kd >3000 nM). Ibrutinib and MT-802 showed differentials in Kd values for the other kinases meeting significance criteria, which correlated nicely with the KINOMEscanTM dataset (Table S3). Additionally, despite the aforementioned lack of ERBB3 degradation, MT802 showed a Kd of 11 nM for this target which was only slightly poorer than ibrutinib’s (2.2 nM). We sought to understand the ability for MT-802 to show reduced inhibition of ITK, JAK3, and MKK7 by structurally aligning the primary sequences of the strongly-inhibited (% of control < 10%) and weakly-inhibited (% of control > 80%) kinase domains (Figure 2C). In line with previous reports, our dataset shows that ibrutinib strongly inhibited many kinases bearing a cysteine homologous to C481. All kinases that were bound significantly by PROTAC and ibrutinib showed complete conservation of the gatekeeper threonine (position 474 in BTK) (Table S2). However, we observed that ITK, JAK3, and MKK7 had a bulky residue (either methionine or phenylalanine) at this gatekeeper site. Structural docking showed that replacement of the threonine with these bulky residues induced significant clashes with the ibrutinib scaffold (Figure 2D). We propose that ibrutinib’s covalent nature can overcome the energy penalty associated with binding


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Biochemistry

these more crowded kinase pockets, but our ibrutinib-based yet reversible PROTAC shows decreased tolerance for the more crowded binding pockets of these off-target kinases, thereby leading to a more restricted substrate set. Of note, there were several other kinases (EGFR, ERBB4, FGFR1/3, TXK, LOK, FLT4, and TNK2) that showed differences in both the KINOMEscanTM and KdELECTTM experiments for MT-802 and ibrutinib engagement levels, but we did not find these to be explicable with the gatekeeper residue hypothesis (Table S2). CSNK1E was the only kinase that met significance criteria for greater binding to MT-802 than ibrutinib and further work will be necessary to both validate and understand the basis of this differential binding. Therefore, while substitution at the gatekeeper position may drive the most apparent class of differentially engaged kinases (ITK, JAK3, and MKK7), there may be other contributing factors that explain the observed differences in MT-802 or ibrutinib binding. When Jurkat cells, a T-lymphocyte cell line, were treated with increasing concentrations of MT-802, we did not observe significant degradation of ITK, likely due to poor ability to bind this kinase (Figure 2E). As our PROTAC is also based on the pomalidomide ligand, we also tested for the degradation of IKZF1 and IKZF3, transcription factors known to be degraded in response to pomalidomide and the related lenalidomide by way of their recruitment to cereblon39. We did not observe MT-802-dependent degradation of either protein when tested in B-lymphocytes derived from CLL patients (Figure 2F). Altogether, these findings demonstrated that degraders based on the noncovalent ibrutinib scaffold may show enhanced specificity for BTK, which in a clinical setting could translate to fewer adverse side-effects from off-target inhibition.


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Figure 2. (A) Results of KINOMEscanTM for selected kinases when screened at 1 µM MT-802 and ibrutinib. Kinases were classified according to the level of inhibition by MT802 (“High” inhibition is a % of control < 10% and “Low” inhibition is a % of control > ACS Paragon Plus Environment

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80%). (B) Bland-Altman difference analysis expressed as a radial bar chart with kinases ordered according to their group. Bars pointing outwards represent kinases inhibited more strongly by ibrutinib than MT-802. Those pointing inwards represent kinases inhibited more strongly by MT-802 than ibrutinib. The shaded gray area represents the interval formed by the 95% limits of agreement (-36.9 to 28.1 % of control). ITK, JAK3, and MKK7 are highlighted as the three kinases for which greatest differential was observed. (C) Kinases were aligned by identity using CLUSTALW algorithm and are grouped according to their level of inhibition by MT-802 (Upper = “high inhibition” and lower = “low inhibition”). Amino acids homologous to positions 481 and 474 in BTK are highlighted in red for all kinases. (D) Crystal structures for ibrutinib in complex with BTK (5P9J), ITK (3QGW), MKK7 (3WZU), and JAK3 (3PJC) were aligned. The space-filling cloud of ibrutinib (purple) is shown to sterically clash with the gatekeeper residues in ITK, MKK7, and JAK3. (E) ITK levels after Jurkat cells (acute T-cell leukemia) were treated with increasing concentrations of MT-802. (F) Primary cells from CLL patients were treated with 1.0 µM lenalidomide and increasing concentrations of MT-802 and levels of IKZF1 and IKZF3 transcription factors were assessed by immunoblotting.

iv. MT-802 Degrades Wild Type and C481S Mutant BTK While we did not observe significant degradation of ERBB3, which possesses a serine at the position homologous to cysteine 481, we were encouraged to see that MT-802 nonetheless retained binding to a kinase with this substitution. This suggested that MT802 has potential to retain interaction with the C481S mutant of BTK, which has been reported in CLL patients exhibiting relapse to ibrutinib therapy17. Relapse is proposed to occur due to loss of the covalent acceptor site, which makes the kinase sensitive only to


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the reversible inhibition provided by ibrutinib, which is at least 40-fold less potent in vitro (Figure 3A). Having already observed potent degradation of wild-type BTK, we proposed that the loss of ibrutinib’s covalent acceptor position would be inconsequential for MT-802’s ability to degrade BTK due to the PROTAC’s need for only a transient association to induce ubiquitination and knockdown. The C481S resistant context, therefore, would serve as an example where the event-driven paradigm of PROTACs can perhaps evade a resistance mechanism arising in response to the occupancyparadigm of inhibitors. When screened for kinase inhibition, the PROTAC and its parent warhead, compound 1, retained inhibition potency against C481S mutant BTK (Figure 3A). Interestingly, the in vitro kinase inhibition assay showed that ibrutinib could still potently inhibit C481S mutant kinase, which is consistent with previous reports57. However, ibrutinib does show at least a 40-fold rightward shift in inhibition potency when the mutation is introduced, unlike MT-802 and compound 1, which highlights the importance of C481 for ibrutinib action (Figure S7). In the in vitro setting, ibrutinib shows nearly 10fold greater inhibition of the mutant kinase than PROTAC, which may be due to an interaction between the backbone amine and carbonyl oxygen of the acrylamide group that is preserved even when serine is substituted (Figure S8). It is important to note that PROTACs are unique in that they often induce protein-protein interactions (between E3 ligase and target protein) of higher affinity than the individual affinities of the PROTAC’s two ligands. Thus, while we may observe a higher IC50 for WT and C481S BTK when the ibrutinib chemotype is incorporated into MT-802, the ternary complex affinity (which can only be measured with cereblon present) may lead to greater levels of inhibition than we might expect from this binary affinity. Since these in vitro assays cannot fully


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recapitulate a cellular context where E3 ligase and other potentially relevant interaction partners are present, we turned to the available cellular systems to study wild-type and C481S BTK signaling. We utilized a previously reported human B-lymphocyte cell line derived from a patient with X-linked agammaglobulinemia (XLA), a primary immunodeficiency caused by inability to produce functional BTK. In this BTK null background, the cells were transduced to express either wild-type or C481S BTK44. In line with our original hypothesis, MT-802 showed equivalent degradation of both wild-type and C481S BTK based both on DC50 and Dmax, the maximal percentage of protein that can be degraded by the PROTAC (Figure 3B). Time course experiments also showed that wild-type and C481S BTK are degraded with similar kinetics (Figure 3C). While the XLA lines also showed that MT-802 reduces the autophosphorylated form of BTK (a marker of active, signaling kinase) concomitant with degradation of the total protein, patient CLL B-cells with a constitutively active BCR pathway reliant on BTK are the ultimate translational tool in studying the ability of this molecule to degrade BTK. To demonstrate the potential clinical applicability of this approach, we turned to isolated primary cells from patients presenting with CLL before and after relapse.


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Figure 3. (A) IC50 values for ibrutinib, SJF-4676, and MT-802 were calculated from 10point dose response curves in duplicate in the presence of 10 µM ATP. (B) Wild-type and C481S BTK-expressing XLA cells were treated with increasing concentrations of


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MT-802 and levels of BTK were quantified by immunoblot. (C) Wild-type and C481S XLA cells were treated with 1 µM MT-802 for indicated times and levels of BTK and pBTK (Y223) were quantified by immunoblot.

v. MT-802 Outperforms Ibrutinib in C481S Primary CLL Patient Samples In order to compare the PROTAC to other BTK-targeting moieties, we studied a range of doses and exposure times of patient cells to MT-802.

Treatment-naïve B-

lymphocytes were isolated from the blood of patients presenting with CLL. Consistent with our experiments with immortalized cell lines, we observed potent knockdown of BTK in the B-lymphocytes of all patients tested (Figure S9). In order to examine the trends of BTK degradation over multiple doses and time points, a mixed effects model was applied to the log-transformed data to estimate differences relative to vehicle or no treatment.

P-values for comparisons have been adjusted using the Dunnett-Hsu

method (for comparisons against vehicle control). Our dose response study shows statistically significant degradation is observed as low as 0.1 µM PROTAC (Figure 4A). Time course experiments showed that maximal degradation was observed between 4 and 12 hours, and the first signs of statistically significant degradation were seen at just 2 hours of treatment (Figure 4B). However, employing a similar rationale as our experiments in NAMALWA, we chose 1.0 µM PROTAC for follow-up experiments because of its ability to induce complete knockdown of BTK as early as 12 hours. Taken together, these experiments confirm the ability of the PROTAC to degrade BTK in isolated patient B-cells. Next, we isolated CLL cells from patients before and after ibrutinib relapse. The emergence of the C481S mutation in BTK, the cause of ibrutinib failure, was confirmed


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by DNA sequencing. MT-802 was able to degrade BTK in both the wild-type baseline and C481S BTK relapsed primary patient samples. Ibrutinib and PROTAC both showed efficacy in inhibiting BTK signaling in baseline CLL cells; however, after relapse, only MT-802 retained its ability to reduce the pool of active, Y223 phosphorylated BTK (Figure 4C). This indicates that reversible binding to C481S BTK is sufficient to induce knockdown when ibrutinib’s scaffold is incorporated into the PROTAC MT-802. Thus, the same chemotype can have very different functional consequences when it is incorporated into molecules following event-driven pharmacology strategy as opposed to an occupancy-driven one. These findings suggest that C481S BTK-expression in CLL will not be wholly sufficient to promote cell survival and proliferation in B-cells treated with our BTK PROTAC.


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Figure 4. (A) Primary cells from patients presenting with CLL were treated with increasing concentrations of MT-802 and levels of BTK were assessed by immunoblot


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(left panel). Results from dose responses in eight independent patients were quantified (right panel). (* corresponds to a p-value

Targeting the C481S Ibrutinib-Resistance Mutation in Bruton's

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