Inhibitor Resistance Mutations Research Articles

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022–23

We evaluated Nanopore sequencing for influenza surveillance. Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. From 941 infections, successful sequencing was achieved in 292/388(75%) available Oxfordshire samples: 231(79%) A/H3N2, 53(18%) A/H1N1, and 8(3%) B/Victoria and in 53/113(47%) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88%); 36/39(92%) Illumina vs. Nanopore comparisons were identical, and 3(8%) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941(90%) Oxfordshire infections were community-acquired. 63/88(72%) potentially healthcare-associated cases shared a hospital ward with ≥1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤5 SNPs, of these, 5(63%) involved potential sources that were also hospital-acquired. Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Ultra-rapid selection of the N74D capsid inhibitor resistance mutation after 3 weeks on lenacapavir

Ultra-rapid selection of the N74D capsid inhibitor resistance mutation after 3 weeks on lenacapavir

Frequency of Fusion Inhibitor Resistance Mutations Among Therapy-Naïve HIV Patients.

Glycoprotein 41 (gp41) of the human immunodeficiency virus type 1 (HIV-1) protein plays a critical role in membrane fusion. Gp41 binds to proteins in the plasma membrane of CD4+ T cells, particularly the T-cell antigen receptor (TCR). These findings indicate that gp41 is involved in the assembly of HIV-1 at the plasma membrane of T cells and affects the stimulation of the TCR. To control HIV-1, new inhibitors were introduced to target the gp41 protein. However, mutations in this region might reduce their efficacy. The Gp41 region was amplified from the sera of 30 patients using nested polymerase chain reaction. The sequences were analyzed by bioinformatics tools to identify mutations and gp41 structural features. Subtyping and the interaction between fusion inhibitors and gp41 proteins were also examined. As the first report from Iran, docking analysis between fusion inhibitors and Iranian gp41 proteins showed that mutations in gp41 could not reduce the efficacy of the fusion inhibitors. Most of the patients were infected with CRF35-AD. Several post-modification positions, including glycosylation and phosphorylation sites, were identified in the gp41 protein. Our findings revealed no known multinational drug resistance to gp41 inhibitors; thus, fusion inhibitors can effectively inhibit HIV in Iranian patients. In addition, the present study introduced a new gp41 region (36-44 aa), which considerably influences the interactions between gp41 inhibitors and the gp41 protein. This region may play a pivotal role in suppressing gp41 inhibitors in CFR35-AD. Furthermore, gp41 can be considered a good target for subtyping analysis via the phylogenetic method.

Prevalence of HIV Drug Resistance Mutations among Treatment-Naive People Living with HIV in a Tertiary Care Center in India.

India has the third-largest number of people living with HIV (PLHIV) in the world. A national program provides free access to standard uniform antiretroviral therapy. However, the program is not monitored by comprehensive drug resistance surveys. The aim of this study was to determine the prevalence of HIV drug resistance mutations (DRMs) among treatment-naive PLHIV in a large antiretroviral treatment center of the national program. This cross-sectional study was done in 2017 and involved 200 consecutive treatment-naive PLHIV. A target fragment of 1,306 bp in the reverse transcriptase and protease regions was amplified. Identification of mutations and drug resistance interpretation was done by HIV Genotypic Resistance Interpretation and International Antiviral Society-USA list. Sequencing was successful in 177 samples. The majority (98.8%; 175/177) belonged to subtype C. Nineteen of 177 patients (10.7%; 95% CI: 6.2%-15.3%) had at least one major DRM. The prevalence of non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations was 10.2% (18/177). The most frequent mutations were E138A/K, A98G, K103N, V179D, and K101H/E. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI) mutations was 1.1% (2/177). None of the samples had major protease inhibitor resistance mutations. The prevalence of NNRTI mutations in this study was >10%, crossing the threshold recommended by the WHO to change the NNRTI-based first-line regimen to non-NNRTI based. In 2021, the national program replaced efavirenz with dolutegravir in the first-line regimen of tenofovir, lamivudine, and efavirenz. As the majority (64%) of PLHIV in India are accessing free ART from the national program, this study highlights the need for regular nationally representative drug resistance surveys for optimizing antiretroviral regimens in the program.

Outcome of Darunavir-Cobicistat-Based Regimens in HIV-Infected People Who Have Experienced Virological Failure.

To evaluate the virological outcome of darunavir-cobicistat (DRVc)-based regimens in adults living with HIV who had experienced virological failure (VF) on any previous drug combination. This was a retrospective cohort study (CSLHIV Cohort) of adults living with HIV who started a DRVc-based regimen with HIV-RNA >50 copies/mL after VF on any previous drug combination. Data on demographics, antiretroviral treatment since HIV diagnosis, and immunological and metabolic parameters from baseline (start of DRVc) to 48 weeks were analyzed in order to assess the cumulative proportion of those who achieved virological success (VS), defined as at least one instance of HIV-RNA <50 copies/mL within 12 months from baseline. Follow-up lasted from the start of the DRVc-based regimen (baseline) to the first instance of HIV-RNA <50 copies/mL, last available visit, or lossto follow-up or death, whichever occurred first. Univariate and multivariate Cox proportional-hazard regression models were used to identify baseline factors associated with VS. A total of 176 individuals were included, and 120 (68.2%) achieved <50 HIV-RNA copies/mL within 12 months since baseline. On multivariate analysis, baseline HDL cholesterol was independently associated with the occurrence of VS (adjusted HR 1.021, 95% CI 1.004-1.038; p=0.014). Among the 120 subjects with VS, 27 (22.5%) had had VF during a median follow-up of 20.8 months since the first undetectable HIV-RNA. Resistance testing after VF was available in two cases, which harboured the HIV variant-bearing protease inhibitor-resistance mutations D30N, I50V, and N88D. During a median follow-up of 38.4 months, 65 of 176 (36.9%) individuals discontinued DRVc for any reason (37 of 120, 30.8%) and achieved VS vs. 28 of 56 (50%) without VS (p=0.019). Time to discontinuation was longer in people with VS (41.5 vs. 23.0 months, p=0.0007). No statistically significant changes were observed in immunological or lipid profiles during follow-up. Most individuals in this study achieved VS within 12 months from the beginning of a DRVc-based regimen; therefore, this treatment represent a viable option for people who have experienced VF on other regimens.

Abstract 1960: CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations

Abstract Bruton’s Tyrosine Kinase (BTK) inhibitor drugs are a mainstay in the treatment of CLL, WM and MCL. They are not curative because of the emergence of BTK inhibitor resistance mutations in as much as 67% of relapsed patients (in CLL) [1]. Frequently observed mutations include BTKC481S, which abrogates covalent binding of irreversible drugs such as acalabrutinib, ibrutinib and zanubrutinib, and BTKT474I and BTKV416L which both abrogate binding of the non-covalent inhibitor drug pirtobrutinib [1, 2]. To combat resistance, third generation BTK inhibitors are needed, which must 1. bind to unique regions in the tumor driving kinases that are not marred by resistance mutations, 2. have sufficient potency for sustained inhibition of BTK signaling, and 3. be sufficiently selective to afford efficacious doses and suitable for a combination regimen. We have developed a platform called Energetically Privileged Ligands (EPriLs), based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket. Their unique binding mode involves connecting the N-terminal and C-terminal lobes of the general kinase fold in a continuous β-sheet, in this way avoiding hotspots for kinase inhibitor resistance mutations. The potent binding of the macrocycle ensures a very long residence time. Using the EPriL platform, we have developed CFON-026, a next generation non-covalent BTK inhibitor with a covalent-like residence time as demonstrated by surface plasmon resonance (SPR) and cellular washout experiments. The biochemical binding KD to BTKWT, determined by SPR, is 0.14 nM. CFON-026 binds mutants with KDs of 0.4 nM (BTKC481S) and and 1.8 nM (BTKT474I). X-ray crystal structures of CFON-026 bound to BTKWT and BTKT474I confirm the unique binding mode and water network around the active site. CFON-026 inhibits the growth of the BTK dependent cell lines TMD8 and Rec-1 with IC50s of 0.45 nM and 1.7 nM, respectively. CFON-026 is very active on TMD8 variants containing endogenous BTK inhibitor resistance mutations (BTKC481S, BTKV416L, BTKT474I and BTKL528W, IC50 range 1-100 nM). CFON-026 has favorable metabolic stability, pharmacokinetic properties, and safety characteristics, and induces complete tumor regression in a TMD8 xenograft model. Since BTK resistance mutations can be detected diagnostically 12-18 months before clinical relapse, CFON-026 could be applied as combination partner to prolong progression free survival on first line BTK inhibitor therapy. Experiments with mixed populations of BTK mutant cell lines, to model a heterogenous tumor, show that CFON-026 can deepen the efficacy of ibrutinib, acalabrutinib and zanubrutinib. In conclusion, CFON-026 is a novel EPriL-based non-covalent BTK inhibitor that could enhance and prolong responses of existing covalent BTK inhibitor therapy. [1] Woyach et al., J Clin Oncol (2017). [2] Wang et al., N. Eng J Med (2022). Citation Format: Jos de Man, Michelle Muller, Joost C.M. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G.T.H. van Mil, Winfried R. Mulder, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1960.

Abstract 6044: EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases

Abstract Heterobifunctional kinase degraders (HKDs) are an important new class of therapeutic modalities, of which the first have recently entered clinical trials. HKDs are small molecules that comprise a kinase-of-interest binding moiety and an E3 ligase binding moiety connected via a spacer. HKDs are excellently suited to target kinases that have a scaffolding function, or that are resistant against conventional inhibitors. A HKD brings a kinase and an E3 ligase in proximity forming a ternary complex leading to ubiquitin-dependent catalytic degradation of the kinase of interest. This ternary complex formation is unique for each kinase which can make HKDs highly specific. To discover novel HKDs, we have developed a platform called Energetically Privileged Ligands (EPriLs), which is based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket with near-covalent affinity, and allows binding to kinases that have acquired known inhibitor resistance mutations. The macrocyclic structure allows attachment of E3 ligase ligands with multiple exit vectors. With the EPriL platform, we have developed novel HKDs for various clinically relevant kinase targets, including Bruton’s Tyrosine Kinase (BTK). BTK inhibitor drugs are used as first line treatment for chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and as second line treatment for mantle cell lymphoma (MCL). Despite their great success in the clinic, many patients on BTK inhibitors relapse eventually, mostly because of the emergence of resistance mutations in the BTK protein. These include BTKC481S, which abrogates binding of covalent drugs, and BTKT474I, BTKL528W and BTKV416L which preclude binding of non-covalent inhibitors like pirtobrutinib. HKDs can combat resistance by degrading such BTK variants, and by removing the scaffold function of BTK. BTK degraders such as NX-5948 and NX-2127 are currently in clinical Phase 1. To show the applicability of the EPriL platform for the discovery of novel BTK degraders, we screened a library of more than 100 EPriL HKDs for biochemical binding to BTK, and tested their propensity to induce degradation of HiBiT-tagged BTKWT, BTKC481S and BTKT474I in HEK293 cell lines. The degrading activity of hits was verified through western blot on the BTK-dependent DLBCL cell line TMD8. The best degraders - based on potency and efficacy - were tested in cell proliferation assays on a panel of TMD8 BTK mutant cell lines. In these lines, endogenous BTK was mutated by CRISPR to BTKC481S, BTKT474I, BTKL528W or BTKV416L. Several BTK degraders had potent (&amp;lt; 20 nM IC50) antiproliferative activity on most TMD8 lines, including difficult-to-target variants such as BTKV416L and BTKL528W. The unique binding mode of the EPriL scaffold thus translates to broad activity on resistance variants, while its selectivity ensures minimal degradation of off-target tyrosine kinases as observed for other degraders. Citation Format: Michelle Muller, Jos de Man, Joost C. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G. van Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6044.

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Portable Nanopore sequencing solution for next-generation HIV drug resistance testing

BackgroundTackling HIV drug resistance is one of major challenges for ending AIDS epidemic, but the elevated expense of cutting-edge genomics hampers the advancement of HIV genotype testing for clinical care. MethodsWe developed a HIV genotype testing pipeline that centers on a cost-efficient portable Nanopore sequencer. Accuracy verification was conducted through comparison with parallel data obtained via fixed-site Pacbio sequencing. Our complete pol-gene sequencing strategy coupled with portable high-throughput sequencing was applied to identify drug resistance mutations across 58 samples sourced from the ART-treated Los Angeles General Medical Center Rand Schrader Clinic (LARSC) cohort (7 samples from 7 individuals) and the ART-naïve Center for HIV/AIDS Vaccine Immunology (CHAVI) cohort (51 samples from 38 individuals). ResultsA total of 472 HIV consensus sequences, each tagged with a unique molecular identifier, were produced from over 1.4 million bases acquired through portable Nanopore sequencing, which matched those obtained independently via Pacbio sequencing. With this desirable accuracy, we first documented the linkage of multidrug cross-resistance mutations across Integrase Strand Transfer inhibitors (INSTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) from an individual failing a second-generation INSTI regimen. By producing more than 500 full-length HIV pol gene sequences in a single portable sequencing run, we detected Protease Inhibitor (PI), Nucleoside Reverse Transcriptase Inhibitor (NRTI), NNRTI and INSTI resistance mutations. All drug resistance mutations identified through portable sequencing were cross-validated using fixed-site Pacbio sequencing. ConclusionsOur accurate and affordable HIV drug resistance testing solution is adaptable for both individual patient care and large-scale surveillance initiatives.

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

Mutation patterns of integrase gene affect antiretroviral resistance in various non-B subtypes of human immunodeficiency virus Type-1 and their implications for patients' therapy.

Although primary integrase strand transfer inhibitor resistance mutations are currently uncommon, the increasing use of integrase strand transfer inhibitor as a key component of the first, second and third-line antiretroviral regimens suggests that the prevalence of integrase drug resistance mutations will likely increase. The rise of several polymorphic mutations and natural polymorphisms also affects the level of susceptibility of human immunodeficiency virus (HIV) type-1 to integrase strand transfer inhibitor. The considerable variability among the various subtypes of human immunodeficiency virus type-1 may contribute to differences in integrase mutations associated with integrase strand transfer inhibitors. Notably, non-B subtypes of HIV type-1 (HIV-1) are the predominant cause of human immunodeficiency virus infection worldwide. The presence of diverse integrase drug resistance mutations can have significant implications on the administration of integrase strand transfer inhibitor-based antiretroviral therapy to patients with human immunodeficiency virus infection.

Human immunodeficiency virus 1 5'-leader mutations in plasma viruses before and after the development of reverse transcriptase inhibitor-resistance mutations.

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) initiation depends on interaction between viral 5'-leader RNA, RT and host tRNA3Lys. Therefore, we sought to identify co-evolutionary changes between the 5'-leader and RT in viruses developing RT-inhibitor resistance mutations. We sequenced 5'-leader positions 37-356 of paired plasma virus samples from 29 individuals developing the nucleoside RT inhibitor (NRTI)-resistance mutation M184V, 19 developing a non-nucleoside RT inhibitor (NNRTI)-resistance mutation and 32 untreated controls. 5'-Leader variants were defined as positions where ≥20 % of next-generation sequencing (NGS) reads differed from the HXB2 sequence. Emergent mutations were defined as nucleotides undergoing a ≥4-fold change in proportion between baseline and follow-up. Mixtures were defined as positions containing ≥2 nucleotides each present in ≥20 % of NGS reads. Among 80 baseline sequences, 87 positions (27.2 %) contained a variant; 52 contained a mixture. Position 201 was the only position more likely to develop a mutation in the M184V (9/29 vs 0/32; P=0.0006) or NNRTI-resistance (4/19 vs 0/32; P=0.02; Fisher's exact test) groups than the control group. Mixtures at positions 200 and 201 occurred in 45.0 and 28.8 %, respectively, of baseline samples. Because of the high proportion of mixtures at these positions, we analysed 5'-leader mixture frequencies in two additional datasets: five publications reporting 294 dideoxyterminator clonal GenBank sequences from 42 individuals and six National Center for Biotechnology Information (NCBI) BioProjects reporting NGS datasets from 295 individuals. These analyses demonstrated position 200 and 201 mixtures at proportions similar to those in our samples and at frequencies several times higher than at all other 5'-leader positions. Although we did not convincingly document co-evolutionary changes between RT and 5'-leader sequences, we identified a novel phenomenon, wherein positions 200 and 201 immediately downstream of the HIV-1 primer binding site exhibited an extraordinarily high likelihood of containing a nucleotide mixture. Possible explanations for the high mixture rates are that these positions are particularly error-prone or provide a viral fitness advantage.

Noncanonical CDK4 signaling rescues diabetes in a mouse model by promoting β cell differentiation.

Expanding β cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain β cell number. β cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of β cell mass in IRS2-deficient mice. Surprisingly, not only β cell mass but also β cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical β cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that β cell mass can be expanded without compromising function.

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

Effect of the L74I Polymorphism on Fitness of Cabotegravir-Resistant Variants of Human Immunodeficiency Virus 1 Subtype A6.

The presence of human immunodeficiency virus (HIV) 1 subtype A6, characterized by the L74I integrase (IN) polymorphism, is associated with confirmed virologic failure in clinical trials of long-acting cabotegravir and rilpivirine. We investigated the effect of L74I on replication capacity (RC) of recombinant viruses carrying this polymorphism in combination with various IN stand-transfer inhibitor resistance mutations. The presence of L74I conferred greater RC to recombinant viruses expressing HIV-1 A6 IN when present together with G118R, G140R, Q148H, and R263K; no significant difference in RC was observed for the Q148K or R mutants. These findings may explain, in part, the association of HIV-1 subtype A6 with virologic failure.

Abstract PR003: Convergent evolution in DNA repair-deficient mCRPC in response to targeted therapy

Abstract BACKGROUND: Distinct metastatic prostate cancer lesions within the same patient can show a range of responses to targeted therapy. These variations in response present a challenge for the interpretation of predictive biomarkers derived from a single lesion. The influence of tumor evolution on how lesions respond to Androgen Signaling Inhibitor (ASI) or PARP inhibitor therapy has been difficulty to study, because few autopsy studies have been performed in heavily treated patients with DNA repair defects. METHODS: In this study, we analyzed an autopsy cohort of 54 tumors from six men with DNA repair-deficient metastatic prostate cancer using whole genome, exome, and targeted DNA sequencing as well as RNA-sequencing. Observations were validated in an independent cohort of 135 mCRPC biopsies with whole genome and transcriptome sequencing. To automate the analysis of mutations conferring resistance to PARP inhibitor therapy, we developed a tool (AARDVARK) that detects and annotates PARP inhibitor resistance mutations. RESULTS: Patients harboring germline inactivation of the DNA repair gene BRCA2 showed consistent BRCA2 biallelic inactivation, while somatic BRCA2 inactivation was not always associated with biallelic deletion and genomic scars of DNA repair inactivation. AARDVARK identified numerous identical resistance mutations in physically distinct tumors in the same individual, consistent with dissemination of these mutations prior to later targeted therapy. We identified DNA structural variations linked to extra-chromosomal DNA (ecDNA) in 52% of mCRPC biopsies, and ecDNA amplified an oncogene 28% of the time. Extensive heterogeneity of structural variants was present at the Androgen Receptor locus, which was the most frequent target of ecDNA amplification (15% of tumors). Cyclins CCND1 and CCNE1 were amplified in 3% of cases, and a long tail of oncogenes were also targeted. Phylogenetic analysis was consistent with AR amplification by ecDNA in response to both androgen deprivation therapy and second-generation ASI. The presence of ecDNA was significantly associated with overall genomic instability, aneuploidy, whole genome doubling, chromothripsis, and with inactivating PTEN alterations. CONCLUSIONS: This study demonstrated that mCRPC lesions continue to evolve on therapy, and their varied responses can be influenced by shared genetic backgrounds. Oncogene amplification by ecDNA is common in mCRPC, particularly at the AR locus, and is linked to genomic instability. Alterations conferring resistance to ASI and PARP inhibitor therapy can be present in individual lesions before metastatic dissemination, and the evolutionary history of tumors plays an important role in their tumor response to targeted therapy. Citation Format: Thaidy Moreno, Meng Zhang, Arian Lundberg, Raunak Shrestha, Martin Sjöström, Anu Pasam, Adam Foye, Anna Trigos, Felix Feng, Shahneen Sandhu, David A. Quigley. Convergent evolution in DNA repair-deficient mCRPC in response to targeted therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR003.

Low level of HIV-1C integrase strand transfer inhibitor resistance mutations among recently diagnosed ART-naive Ethiopians

With the widespread use of Integrase strand transfer inhibitors (INSTIs), surveillance of HIV-1 pretreatment drug resistance is critical in optimizing antiretroviral treatment efficacy. However, despite the introduction of these drugs, data concerning their resistance mutations (RMs) is still limited in Ethiopia. Thus, this study aimed to assess INSTI RMs and polymorphisms at the gene locus coding for Integrase (IN) among viral isolates from ART-naive HIV-1 infected Ethiopian population. This was a cross-sectional study involving isolation of HIV-1 from plasma of 49 newly diagnosed drug-naive HIV-1 infected individuals in Addis-Ababa during the period between June to December 2018. The IN region covering the first 263 codons of blood samples was amplified and sequenced using an in-house assay. INSTIs RMs were examined using calibrated population resistance tool version 8.0 from Stanford HIV drug resistance database while both REGA version 3 online HIV-1 subtyping tool and the jumping profile Hidden Markov Model from GOBICS were used to examine HIV-1 genetic diversity. Among the 49 study participants, 1 (1/49; 2%) harbored a major INSTIs RM (R263K). In addition, blood specimens from 14 (14/49; 28.5%) patients had accessory mutations. Among these, the M50I accessory mutation was observed in a highest frequency (13/49; 28.3%) followed by L74I (1/49; 2%), S119R (1/49; 2%), and S230N (1/49; 2%). Concerning HIV-1 subtype distribution, all the entire study subjects were detected to harbor HIV-1C strain as per the IN gene analysis. This study showed that the level of primary HIV-1 drug resistance to INSTIs is still low in Ethiopia reflecting the cumulative natural occurrence of these mutations in the absence of selective drug pressure and supports the use of INSTIs in the country. However, continues monitoring of drug resistance should be enhanced since the virus potentially develop resistance to this drug classes as time goes by.

Dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF-mutant solid tumors.

Combination BRAF and MEK inhibitor therapy is an active regimen in patients who have BRAF V600E-mutated tumors; however, the clinical efficacy of this therapy is limited by resistance. Preclinically, the addition of heat shock protein 90 (HSP90) inhibition improves the efficacy of BRAF inhibitor therapy in both BRAF inhibitor-sensitive and BRAF inhibitor-resistant mutant cell lines. Cancer Therapy Evaluation Program study 9557 (ClinicalTrials.gov identifier NCT02097225) is a phase 1 study that was designed to assess the safety and efficacy of the small-molecule HSP90 inhibitor, AT13387, in combination with dabrafenib and trametinib in BRAF V600E/K-mutant solid tumors. Correlative analyses evaluated the expression of HSP90 client proteins and chaperones. Twenty-two patients with metastatic, BRAF V600E-mutant solid tumors were enrolled using a 3+3 design at four dose levels, and 21 patients were evaluable for efficacy assessment. The most common tumor type was colorectal cancer (N=12). Dose-limiting toxicities occurred in one patient at dose level 3 and in one patient at dose level 4; specifically, myelosuppression and fatigue, respectively. The maximum tolerated dose was oral dabafenib 150mg twice daily, oral trametinib 2mg once daily, and intravenous AT13387 260mg/m2 on days 1, 8, and 15. The best response was a partial response in two patients and stable disease in eight patients, with an overall response rate of 9.5% (90% exact confidence interval [CI], 2%-27%), a disease control rate of 47.6% (90% CI, 29%-67%), and a median overall survival of 5.1months (90% CI, 3.4-7.6 months). There were no consistent proteomic changes associated with response or resistance, although responders did have reductions in BRAF expression, and epidermal growth factor receptor downregulation using HSP90 inhibition was observed in one patient who had colorectal cancer. HSP90 inhibition combined with BRAF/MEK inhibition was safe and produced evidence of modest disease control in a heavily pretreated population. Additional translational work may identify tumor types and resistance mechanisms that are most sensitive to this approach.

Abstract 2850: NX-5948 promotes selective, sub-nanomolar degradation of inhibitor-resistant BTK mutants

Abstract Small molecule kinase inhibitors have revolutionized the treatment of hematological malignancies by suppressing signaling pathways essential for tumor cell survival. Bruton’s Tyrosine Kinase (BTK) inhibitors are widely used in the clinic for treatment of patients with B cell malignancies. Acquired resistance mutations, however, can reduce or eliminate their efficacy and represent a growing challenge. Mutations at C481 dramatically reduce the binding of covalent BTK inhibitors, whereas other clinically-observed mutations such as V416L, T474I, and L528W reduce or eliminate the activity of next-generation non-covalent inhibitors. Some of these mutations abolish BTK kinase activity while retaining intact BCR signaling and BTK-dependent growth, indicating that mutant BTK elicits scaffold-mediated signaling essential for malignant B cell survival. To assess the impact of resistance mutations on the activity of BTK inhibitors, we generated a DLBCL line (TMD8) harboring BTK-C481S, V416L, T474I, or L528W point mutations. The C481S mutation eliminated the anti-proliferative effects of covalent inhibitors ibrutinib, acalabrutinib, and zanubrutinib, while the V416L, T474I, and L528W mutations dramatically reduced the activity of pirtobrutinib, vecabrutinib, and fenebrutinib. L528W largely abolished the activity of ibrutinib and zanubrutinib, whereas V416L substantially reduced the activity of acalabrutinib. This variability in BTK inhibitor sensitivity to resistance mutations complicates treatment decisions for patients who relapse on BTK inhibitors, necessitating agents that can more broadly target resistance mutations. Here we assessed the activity of NX-5948, a heterobifunctional degrader molecule that induces the targeted degradation of BTK. We used surface plasmon resonance to evaluate the binding of NX-5948 to WT and mutant (C481S, T474I, V416L and L528W) BTK proteins. NX-5948 binds potently to BTK WT, C481S and T474I with single-digit nanomolar affinities, but loses potency against the V416L (14-fold) and L528W (33-fold) mutants. Despite this reduction in binary binding affinity, NX-5948 induces sub-nanomolar degradation of all mutant forms of BTK and potently suppresses expression of activation markers and proliferation in TMD8 cells harboring these mutations. We propose that the positive cooperativity that NX-5948 induces between BTK and the E3 ligase cereblon contributes to its potent and sustained degradation activity against BTK resistance mutants. We also assessed the selectivity of NX-5948 by global proteomics and observed exquisite selectivity across cell types and conditions. The exceptional potency, selectivity, and activity of NX-5948 against BTK mutants warrant its investigation in clinical settings that develop diverse inhibitor resistance. A phase 1a/b trial of NX-5948 for patients with relapsed or refractory B-cell malignancies is ongoing (NCT05131022). Citation Format: Mark Andrew Noviski, Nivetha Brathaban, Ratul Mukerji, Stephanie Yung, Jordan Ye, Hugo Bousquet, Mateo Sanchez Garcia de los Rios, Brandon Bravo, Jiwen Chen, Paul Auger, Jeffrey Mihalic, Hao Lu, Cristiana Guiducci, Gwenn Hansen. NX-5948 promotes selective, sub-nanomolar degradation of inhibitor-resistant BTK mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2850.