Mutant Variants Research Articles

Tools for editing the mammalian mitochondrial genome.

The manipulation of animal mitochondrial genomes has long been a challenge due to the lack of an effective transformation method. With the discovery of specific gene editing enzymes, designed to target pathogenic mitochondrial DNA mutations (often heteroplasmic), the selective removal or modification of mutant variants has become a reality. Because mitochondria cannot efficiently import RNAs, CRISPR has not been the first choice for editing mitochondrial genes. However, the last few years witnessed an explosion in novel and optimized non-CRISPR approaches to promote double-strand breaks or base-edit of mtDNA in vivo. Engineered forms of specific nucleases and cytidine/adenine deaminases form the basis for these techniques. I will review the newest developments that constitute the current toolbox for animal mtDNA gene editing in vivo, bringing these approaches not only to the exploration of mitochondrial function, but also closer to clinical use.

Real-time PCR detection of PI*S and PI*Z alleles of SERPINA1 gene using SYBR green

BackgroundAlpha-1 antitrypsin deficiency is an underdiagnosed genetic condition that predisposes to pulmonary complications and is mainly caused by rs28929474 (PI*Z allele) and rs17580 (PI*S allele) mutations in the SERPINA1 gene. ObjectiveDevelopment of a homogeneous genotyping test for detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye. MethodsSixty individuals, which included all possible genotypes that result from combinations of rs28929474 and rs17580 single nucleotide variants, were assayed with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine. ResultsA clear discrimination of mutant and wild-type variants was achieved in the genetic loci that define PI*S and PI*Z alleles. Specific amplicons showed a difference of 2.0 °C in melting temperature for non-S and S variants and of 2.9 °C for non-Z and Z variants. ConclusionsThe developed genotyping method is robust, fast, and easily scalable on a standard real-time PCR platform. While it overcomes the handicaps of non-homogeneous approaches, it greatly reduces genotyping costs compared with other homogeneous approaches.

Characteristic fingerprint spectrum of α-synuclein mutants on terahertz time-domain spectroscopy

α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is involved in the pathogenesis of Parkinson’s disease. Point mutations and multiplications of α-synuclein (A30P and A53T) are correlated with early-onset Parkinson’s disease characterized by rapid progression and poor prognosis. Currently, the clinical identification of SNCA variants, especially disease-related A30P and A53T mutants, remains challenging and also time consuming. This study aimed to develop a novel label-free detection method for distinguishing the SNCA mutants using transmission terahertz (THz) time-domain spectroscopy. The protein was spin-coated onto the quartz to form a thin film, which was measured using THz time-domain spectroscopy. The spectral characteristics of THz broadband pulse waves of α-synuclein protein variants (SNCA wild type, A30P, and A53T) at different frequencies were analyzed via Fourier transform. The amplitude A intensity (AWT, AA30P, and AA53T) and peak occurrence time in THz time-domain spectroscopy sensitively distinguished the three protein variants. The phase φ difference in THz frequency domain followed the trend of φWT > φA30P > φA53T. There was a significant difference in THz frequency amplitude A′ corresponding to the frequency ranging from 0.4 to 0.66 THz (A′A53T > A′A30P > A′WT). At a frequency of 0.4–0.6 THz, the transmission T of THz waves distinguished three variants (TA53T > TA30P > TWT), whereas there was no difference in the transmission T at 0.66 THz. The SNCA wild-type protein and two mutant variants (A30P and A53T) had distinct characteristic fingerprint spectra on THz time-domain spectroscopy. This novel label-free detection method has great potential for the differential diagnosis of Parkinson’s disease subtypes.

Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4.

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.

Abstract ND03: Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor

Abstract RASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The RASG12D mutation increases the abundance of the active, GTP-bound state of RAS (RASG12D(ON)) and occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of RAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. RMC-6236, an investigational RASMULTI(ON) inhibitor currently in clinical testing, selectively targets the active, GTP-bound state of both mutant and wild-type RAS variants, including RASG12D(ON) and has shown clinical anti-tumor activity against tumors harboring KRASG12D. Efforts to find a mutant-selective RASG12D(ON) inhibitor led to the discovery of RMC-9805.The investigational agent RMC-9805 is a first-in-class, orally bioavailable, mutant selective covalent inhibitor of RASG12D(ON) that forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the “ON” state of RASG12D, enabling selective covalent engagement of Asp-12 and disrupting downstream RAS signaling by steric occlusion of effector binding. In contrast to other examples in the literature of covalent inhibitors of KRASG12D that rely on highly reactive, low stability warheads to overcome the low intrinsic reactivity of aspartic acid, RMC-9805 employs a warhead with low intrinsic reactivity and high stability under biological conditions. Rapid, selective, covalent modification of RASG12D by RMC-9805 is enabled by our tri-complex technology. We used structure-guided design to position the warhead in the CypA-RAS interface in an optimal orientation that enhanced crosslinking of Asp-12 allowing us to use a warhead that showed no intrinsic reactivity to a model aspartic acid system and was stable to enable oral dosing. RMC-9805 drove selective and persistent covalent modification of KRASG12D in human cancer cell lines in vitro, leading to deep and durable suppression of RAS pathway activity, inhibition of cell proliferation, and apoptosis. RMC-9805 monotherapy induced tumor regressions at well-tolerated doses in a majority of preclinical PDAC and NSCLC models harboring KRASG12D. Though a more heterogeneous response was observed in KRASG12D CRC models, combinations of RMC-9805 with either RMC-6236 (RASMULTI(ON) inhibitor) or an anti-EGFR antibody improved the depth of response and delayed the onset of resistance in vivo. In addition, RMC-9805 promoted cancer-associated neoantigen recognition and synergized with immunotherapy in preclinical models. RMC-9805 is also CNS penetrant and drove regressions in intracranial xenograft models of human KRASG12D PDAC. RMC-9805 is a first-in-class orally bioavailable, mutant selective and covalent RASG12D inhibitor currently in Phase 1 clinical trial (NCT06040541). Citation Format: John E. Knox, G. Leslie Burnett, Caroline Weller, Lingyan Jiang, Dongyu Zhang, Nicole Vita, Abby Marquez, Kyle J. Seamon, Andrea Gould, Marie Menard, Elsa Quintana, Zhe Chen, Zhican Wang, Zhengping Wang, Elena S. Koltun, Malika Singh, Jingjing Jiang, David Wildes, Jacqueline A.M. Smith, Adrian L. Gill. Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND03.

Diverse pro-inflammatory ability of mutated spike protein derived from variant strains of SARS-CoV-2

The severity of COVID-19 has been reported to differ among SARS-CoV-2 mutant variants. The overactivation of macrophages is involved in severe COVID-19, yet the effects of SARS-CoV-2 mutations on macrophages remain poorly understood. To clarify the effects, we examined whether mutations of spike proteins (S-proteins) affect macrophage activation. CD14+ monocyte-derived macrophages were stimulated with the recombinant S-protein of the wild-type, Delta, and Omicron strains or live viral particles of individual strains. Regarding IL-6 and TNF-α, Delta or Omicron S-protein had stronger or weaker pro‑inflammatory ability, respectively, than the wild-type. Similar trends were observed between S-proteins and viral particles. S-protein mutations could be related to the diversity in macrophage activation and severity rates in COVID-19 caused by various SARS-CoV-2 strains.

Abstract 4684: A comprehensive platform for identification of KRAS-specific synthetic lethal targets using patient-derived cells

Abstract Colorectal cancer (CRC) remains a global health concern, constituting a large portion of cancer incidence and mortality. Despite advances in comprehending the genetic and epigenetic landscape of CRC and progress achieved in developing KRAS-specific inhibitors poised for clinical evaluation, a significant unmet medical need persists. This underscores the necessity for innovative therapeutic interventions in this field, as effective therapeutic strategies targeting specific molecular vulnerabilities are currently lacking. Our study aims to address this gap by focusing on the identification of synthetic lethal (SL) inhibitors specific to the KRAS pathway. We have established a platform utilizing human intestinal stem cell (hISC)-derived CRC model cells, patient-derived xenografts (PDXs), and clinical samples for genomic and functional analyses. Employing CRISPR/Cas9 technology, we introduced common CRC mutations into hISCs, generating isogenic CRC model cells with diverse mutational backgrounds including KRAS G12D. Additionally, we developed PDXs from CRC patients and collected biopsy samples from primary and metastatic tumors. Through RNA-seq and whole-exome sequencing (WES), we identified molecular signatures and pathways associated with different mutational variants. Our innovative platform enables high-throughput screening, incorporating both small molecule screening and CRISPR/Cas9 dropout screening with a genome-wide library on CRC model cells. Notably, our focus on KRAS-specific SL inhibitors distinguishes our approach, aiming for targeted therapies tailored to specific CRC subtypes. Validation of driver mutations, such as APC, KRAS, and TP53, in patient-derived material showcased the relevance of our model. High-quality screening results, meeting industry-standard QC criteria, allowed us to identify essential and tumor suppressor genes critical for CRC. Importantly, the use of normal hISCs enabled the identification of genes crucial only for transformed cells. The platform serves as a powerful tool for target discovery and validation in CRC, emphasizing the significance of primary material-derived cells. Beyond CRC, our findings have broader implications for other cancer types and personalized medicine approaches. By focusing on the identification of KRAS-specific SL inhibitors, our research contributes to the development of novel therapies for CRC, addressing the urgent need for targeted interventions in this heterogeneous malignancy. Citation Format: Marcin Dulęba, Eliza Zimoląg, Joanna Szuszkiewicz, Marcin Serocki, Joanna Szklarczyk, Olga Dracz, Zuzanna Kurzejamska, Diego Coelho, Michał Combik, Oleksii Bryzghalov, Joanna Krawczyk, Agata Stachowicz, Michał Mikula, Krzysztof Brzózka, Rafał Dziadziuszko, Tomasz Rzymski, Andrzej Mazan. A comprehensive platform for identification of KRAS-specific synthetic lethal targets using patient-derived cells [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 4684.

Abstract 6045: Development and characterization of ATC-324: An AUTOTAC-based androgen receptor degrader for prostate cancer treatment

Abstract Prostate cancer (PCa) progression is largely driven by the androgen receptor (AR), making it a prime target for therapy. However, therapy resistance often arises due to AR mutations and splice variants, such as AR-v7. AUTOTAC (AUTOphagy-TArgeting Chimera) is a novel protein degradation platform that utilizes the autophagy-lysosomal pathway to selectively degrade disease-causing proteins. In this study, we characterize ATC-324, an AUTOTAC-based AR degrader designed for the treatment of PCa. ATC-324 comprises enzalutamide linked to YT 6-2, a small molecule activator of the autophagy receptor p62/SQSTM1. ATC-324 induces the formation of the AR/p62 complex while activating autophagic flux, leading to the autophagy-lysosomal degradation of AR. To characterize ATC-324, we employed various AR-dependent, AR-null, and enzalutamide-resistant PCa cell lines. Given that bone metastasis is a major clinical complication of castrate-resistant PCa associated with greater morbidity and mortality, we utilized the bone-in-culture array (BICA) assay, an ex vivo bones culture model, to investigate the impact of ATC-324 on PCa cell survival and growth in the bone microenvironment. We demonstrated that ATC-324 effectively degrades wild-type, mutant, and splice variants of AR. In addition, ATC-324 induces AR-dependent apoptosis and cytotoxicity. Importantly, ATC-324 maintains its potency in enzalutamide-resistant PCa cells. Utilizing the BICA assay, we showed that ATC-324 exhibits cytotoxicity against 22Rv1 bone micrometastases at even lower concentrations than in 2D cell culture. In conclusion, our study demonstrates the successful development and characterization of ATC-324 as a potent and selective degrader of various forms of AR. Furthermore, its effectiveness against enzalutamide-resistant cells indicates its capability to overcome therapy resistance. The observed cytotoxicity against bone micrometastases suggests a therapeutic potential for combating bone metastasis, a major complication of castration-resistant PCa. Our study underscores the potential of the AUTOTAC platform to selectively degrade disease-causing proteins Citation Format: Tri Pham, Tae Hyun Bae, Ki Woon Sung, Abdo Najy, Alaleh Zamiri, Hyejeong Jang, Su Ran Mun, Seongho Kim, Dongping Shi, Steven Kregel, Elizabeth Heath, Michael Cher, Yong Tae Kwon, Hyeong-Reh Kim. Development and characterization of ATC-324: An AUTOTAC-based androgen receptor degrader for prostate cancer treatment [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 6045.

Abstract 6585: Combination RASG12C(ON) and RASMULTI(ON) inhibition overcomes resistance and prolongs durability in preclinical models of mutant KRASG12C-driven cancers

Abstract Inhibitors of the GDP-bound state of mutant KRASG12C protein (KRASG12C(OFF)), e.g., adagrasib and sotorasib, have shown clinical activity in patients with KRASG12C-driven NSCLC, CRC and PDAC. However, most patients exhibit progression on monotherapy treatment in months, underlining the need for combination therapeutic approaches that improve the durability of the anti-tumor activity seen with KRASG12C inhibitors. RMC-6291 is a mutant-selective covalent inhibitor of the GTP-bound RAS(ON) form of RASG12C, while RMC-6236 is a non-covalent RASMULTI(ON) inhibitor of both mutant and wild-type variants of canonical RAS isoforms. In models harboring diverse alterations driving resistance mechanisms to KRASG12C(OFF) inhibitors – including reactivation of KRASG12C and wild-type RAS by increased RTK signaling, acquired secondary oncogenic RAS mutations, and KRAS amplification – both RMC-6291 and RMC-6236 exhibited superior anti-cancer activity as single agents when compared to KRASG12C(OFF) inhibitors. Thus, we hypothesized that a combination of RMC-6291 and RMC-6236 could address a broad spectrum of observed clinically relevant resistance mechanisms to KRASG12C(OFF) inhibitors that reactivate RAS signaling via mutant and wild-type RAS isoforms and compromise the depth and durability of anti-tumor response. In a mouse clinical trial with KRASG12C xenograft models, daily oral administration of RMC-6291 induced objective responses in 18 out of 25 NSCLC PDX and CDX models as assessed by mRECIST. In a subpanel of 7 NSCLC models that are relatively refractory to KRASG12C(OFF) inhibitor monotherapy, RMC-6236 alone somewhat prolonged the durability of response, while the combination of RMC-6291 with RMC-6236 improved the depth of response and significantly extended the durability thereof. In contrast to NSCLC, in models of KRASG12C CRC tumors a more heterogeneous response to RMC-6291 monotherapy was observed with objective responses in 4 out of 15 CRC models. The addition of RMC-6236 to RMC-6291 substantially improved the depth and duration of anti-tumor response in the resistant models to KRASG12C inhibitor alone. The combination of RMC-6291 with RMC-6236 at preclinical target doses did not affect the systemic or tumor exposures of each compound and was well tolerated in tumor-bearing immune-deficient animals. Both investigational agents RMC-6236 and RMC-6291 have shown promising anti-tumor activity as monotherapies in preclinical models and, more recently, showed preliminary clinical activity in patients with RAS-driven tumors at exposures that were well tolerated. The addition of RMC-6236 to RMC-6291 shows combinatorial activity in preclinical models that are relatively less responsive to either inhibitor alone. A clinical trial combining RMC-6291 with RMC-6236 in patients with mutant KRASG12C tumors is currently enrolling. Citation Format: Xing Wei, Matthew Holderfield, Yu Chi Yang, Kyle Seamon, Jay Dinglasan, Nilufar Montazar, Bianca Lee, Shurui Cai, Lick Lai, Ida Aronchik, Stephanie Chang, Zhican Wang, Pete Wildes, Jacqueline A. Smith, Jingjing Jiang, Mallika Singh. Combination RASG12C(ON) and RASMULTI(ON) inhibition overcomes resistance and prolongs durability in preclinical models of mutant KRASG12C-driven cancers [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 6585.

Abstract 3993: A tunable safety switch for solid tumor CAR T-cell therapy

Abstract On-target off-tumor toxicity, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS) are severe immune-related adverse events (irAEs) that are frequently associated with Chimeric Antigen Receptor (CAR) T-cell therapy. Current efforts to manage such therapy-related toxicities involve incorporation of an inducible suicide agent within CAR constructs, such as iCaspase-9 or herpes simplex virus type 1 thymidine kinase that can be selectively activated to produce toxic effects within CAR T cells and attenuate their activity. However, while activation of these agents helps to mitigate or overcome such unwarranted toxicities, the therapeutic benefit of CAR T cells anti-tumor activity is also compromised. Therefore, to continue maintenance of CAR T cells’ therapeutic function while minimizing irAEs, an ideal safety switch should 1) rapidly inhibit the activation and proliferation of CAR T cells exposed to the target antigen, 2) reversibly inhibit activity without inducing CAR T cell elimination and 3) be clinically translatable for safe application in patients. Our laboratory investigated one such safety switch to inhibit CAR T-cell activity while maintaining their therapeutic function. We showed that incorporating a mutant variant of c-KIT D816V (KITv) in the intracellular domain of mesothelin-targeting second-generation CAR T cells (M28z-KITv) improved efficacy in solid tumors with low antigen, or an immunosuppressive environment. Herein, we evaluate the use of Dasatinib, a clinically available multitarget (BCR, SRC, c-KIT) tyrosine kinase inhibitor (TKI), as a tunable safety switch to reversibly inhibit M28z-KITv CAR T-cell functional activity. In cohorts of mice established with lung adenocarcinoma, daily administration of Dasatinib starting on day 1 or day 3 after CAR T-cell administration stabilized tumor growth, which otherwise continued to regress in untreated mice, indicating inhibition of CAR T-cell functional activity. Upon discontinuation of Dasatinib, tumors regressed, indicating reversal of CAR T-cell functional activity. In an experiment conducted to investigate functional persistence of CAR T cells upon long-term exposure to Dasatinib (1 month), we noted uninhibited activity of CAR T cells to rechallenged tumors. Dasatinib, thus may act as a tunable safety switch to regulate M28z-KITv CAR T-cell activity without compromising its therapeutic function. Citation Format: Kyohei Misawa, Meriem Taleb, Srijita Banerjee, William-Ray Vista, Navin K. Chintala, Prasad S. Adusumilli. A tunable safety switch for solid tumor CAR T-cell therapy [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 3993.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma [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 1578.

Abstract 1927: Resistance to RAS-GTP inhibition in models of pancreatic ductal adenocarcinoma arises downstream of RAS effectors

Abstract More than 90% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS that drive it into an active, GTP-bound state (KRAS(ON)), producing excessive signaling via MAPK and other effector pathways. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). Exposure to RMC-7977 suppressed the MAPK biomarker p-ERK, reduced cell growth, and induced apoptosis in multiple human RAS-addicted cancer cell lines. Assessment of RMC-7977 in a series of PDAC preclinical model systems revealed broad anti-tumor activity and treatment was well tolerated at translatable doses. The K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) autochthonous mouse model was utilized to study the effects of Ras-GTP inhibition in both tumor and normal tissues. At doses that drove anti-tumor activity, RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissue, with full pathway inhibition at 4 h post treatment that was restored by 24 h. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. RMC-7977 treatment drove regressions and an unprecedented ~3-fold extension of overall survival in KPC mice. After an initial response, all tumors eventually developed resistance on treatment, affording the opportunity to study acquired resistance mechanisms to RAS-GTP inhibition. Strikingly, in all resistant tumors, acute inhibition of MAPK and PI3K signaling was still apparent following treatment. These results indicate that RAS signaling re-activation mechanisms that have been reported as clinical mechanisms of resistance to mutant-selective KRASG12C inhibitors (e.g. second-site mutations or upregulation of upstream RTK) may be insufficient to confer resistance to a compound that inhibits all RAS isoforms. Rather, in this model we observed activation of alternative oncogenic mechanisms downstream of RAS effector signaling, including frequent focal amplification of Myc, focal amplification of Jun, and/or activation of Yap/Taz/Tead signaling in most resistant KPC tumors. Together these results demonstrate preclinical activity of RMC-7977 at tolerable doses, producing unprecedented responses even in highly chemoresistant PDAC models such as the KPC mouse. The observed range of potential resistance mechanisms suggests that RAS-GTP inhibition imposes a narrow evolutionary path to the development of acquired resistance in this context. These preclinical findings may help inform the ongoing development of investigational RAS inhibitors and the design of potential combinatorial treatment strategies to forestall resistance. Citation Format: Urszula N. Wasko, Jingjing Jiang, Yingyun Wang, Lorenzo Tomassoni, Lingyan Jiang, Marie Menard, Alvaro Curiel-Garcia, Tanner C. Dalton, Margo Orlen, Cole Edwards, Clint Stalnecker, Julien Dilly, Stephanie Chang, Stephen A. Sastra, Carmine F. Palermo, Timour Baslan, Sha Tian, Matthew Holderfield, Elsa Quintana, Zhengping Wang, Jacqueline A. Smith, Andrea Califano, David Wildes, Andrew J. Aguirre, Robert H. Vonderheide, Ben Z. Stanger, Scott W. Lowe, Channing J. Der, Mallika Singh, Kenneth P. Olive. Resistance to RAS-GTP inhibition in models of pancreatic ductal adenocarcinoma arises downstream of RAS effectors [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 1927.

Abstract 3874: Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues, with full pathway inhibition at 4 hours post treatment that was restored by 24 hours. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. To understand the dynamic response of malignant cells following RAS-GTP inhibition, we did single cell RNA sequencing on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms [Maroling., et al. 2006; Alvarez., et al. 2016], we performed regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of >5000 transcriptional regulatory proteins per cell. Additionally, we assessed the impact of treatment on malignant cell subtypes as previously identified by our team based on their sets of hyperactivated developmental transcription factors [Laise., et al. 2022]. As expected, RAS pathway transcription factors, such as FOS and JUN, were repressed relative to controls in malignant cells by 4 hours after treatment with RMC-7977. However, at 24 hours, responses varied by cellular subtype: well-differentiated Gastrointestinal Lineage State (GLS) cells had restored MAPK activity, while poorly differentiated Morphogenic State (MOS) cells were still inactivated. Strikingly, we found that after one week of treatment (in actively regressing tumors, representing a state of residual disease), nearly all malignant cells had assumed the GLS state. Acquisition of resistance after weeks or months of treatment was associated with a restored diversity of malignant cell states, suggesting that the GLS state serves as a regulatory “safe harbor” in which residual malignant cells can undergo evolution and adaptation. Mechanistically, we found that RAS pathway signaling directly activates transcriptional regulators that drive the MOS state, elucidating an early adaptive response to RAS-GTP inhibition that enables residual cells to survive treatment prior to development of acquired resistance. Citation Format: Lorenzo Tomassoni, Urszula N. Wasko, Alvaro C. Garcia, Tanner C. Dalton, Pasquale Laise, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Adaptive heterogeneity enables the survival of residual malignant PDAC cells in response to RAS-GTP inhibition [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 3874.

The zinc finger motif in the mitochondrial large ribosomal subunit protein bL36m is essential for optimal yeast mitoribosome assembly and function

Ribosomes across species contain subsets of zinc finger proteins that play structural roles by binding to rRNA. While the majority of these zinc fingers belong to the C2-C2 type, the large subunit protein L36 in bacteria and mitochondria exhibits an atypical C2-CH motif. To comprehend the contribution of each coordinating residue in S. cerevisiae bL36m to mitoribosome assembly and function, we engineered and characterized strains carrying single and double mutations in the zinc coordinating residues. Our findings reveal that although all four residues markedly influence protein stability, C to A mutations in C66 and/or C69 have a more pronounced effect compared to those at C82 and H88. Importantly, protein stability directly correlates with the assembly and function of the mitoribosome and the growth rate of yeast in respiratory conditions. Mass spectrometry analysis of large subunit particles indicates that strains deleted for bL36m or expressing mutant variants have defective assembly of the L7/L12 stalk base, limiting their functional competence. Furthermore, we employed a synthetic bL36m protein collection, including both wild-type and mutant proteins, to elucidate their ability to bind zinc. Our data indicate that mutations in C82 and, particularly, H88 allow for some zinc binding albeit inefficient or unstable, explaining the residual accumulation and activity in mitochondria of bL36m variants carrying mutations in these residues. In conclusion, stable zinc binding by bL36m is essential for optimal mitoribosome assembly and function. MS data are available via ProteomeXchange with identifierPXD046465.

MicroRNA-7 attenuates secondary brain injury following experimental intracerebral hemorrhage via inhibition of NLRP3

Background and purposeThe pathophysiological mechanisms underlying brain injury resulting from intracerebral hemorrhage (ICH) remain incompletely elucidated, and efficacious therapeutic interventions to enhance the prognosis of ICH patients are currently lacking. Previous research indicates that MicroRNA-7 (miR-7) can suppress the expression of Nod-like receptor protein 3 (NLRP3), thereby modulating neuroinflammation in Parkinson's disease pathogenesis. However, the potential regulatory effects miR-7 on NLRP3 inflammasome after ICH are yet to be established. This study aims to ascertain whether miR-7 mitigates secondary brain injury following experimental ICH by inhibiting NLRP3 and to investigate the underlying mechanisms. MethodsAn ICH model was established by stereotaxically injecting 100 μL of autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. Subsequently, these rats were allocated into three groups: sham, ICH + Vehicle, and ICH + miR-7, each comprising 18 animals. Twelve hours post-modeling, rats received intraventricular injections of 10 μL physiological saline, 10 μL phosphate, and 10 μL phosphate-buffered saline solution containing 0.5 nmol of miR-7 mimics, respectively. Neurological function was assessed on day three post-modeling, followed by euthanasia for brain tissue collection. Brain water content was determined using the dry-wet weight method. The expression of inflammatory cytokines in cerebral tissues surrounding the hematoma was analyzed through immunohistochemistry and Western blot assays. These cytokines were re-evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, bioinformatics tools were employed to predict miR-7′s binding to NLRP3. A wild-type luciferase reporter gene vector and a corresponding mutant vector were constructed, followed by transfection of miR-7 mimics into HEK293T cells to assess luciferase activity. ResultsOur study demonstrates that the administration of miR-7 mimics markedly reduced neurological function scores and attenuated brain edema in rats following ICH. A significant upregulation of NLRP3 expression in microglia/macrophage adjacent to the hematoma was observed, substantially reduced after the treatment with miR-7 mimics. Furthermore, this intervention ameliorated neurodegenerative changes and effectively decreased the protein and mRNA levels of pro-inflammatory cytokines, namely TNF-α, IL-1β, IL-6, and Caspase1, in the cerebral tissues proximate to the hematomas. In addition, miR-7 mimics distinctly inhibited the luciferase activity associated with the wild-type reporter gene, an effect not mirrored in its mutant variant. ConclusionsThe miR-7 suppressed NLRP3 expression in microglia/macrophage to reduce the production of inflammatory cytokines, leading to conducting certain neuroprotection post-ICH in rats.

222P Mutational variant allele frequency profile as a biomarker of response to immune checkpoint inhibitors in non-small cell lung cancer

222P Mutational variant allele frequency profile as a biomarker of response to immune checkpoint inhibitors in non-small cell lung cancer

Unveiling Potential Therapies: Molecular Docking Analysis of CAMKK2 and Its Mutant Variants with CAMKK2 Inhibitors in Indonesian Patients with HIV-Sensory Neuropathy

Unveiling Potential Therapies: Molecular Docking Analysis of CAMKK2 and Its Mutant Variants with CAMKK2 Inhibitors in Indonesian Patients with HIV-Sensory Neuropathy

Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes.

Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants.

GATA2 mutant variant allele frequency may reflect prognosis in Chinese adult patients with de novo cytogenetically normal acute myeloid leukemia.

Exploration of variant allele frequency (VAF) of GATA2 mutations (GATA2mut) provides insights into acute myeloid leukemia (AML) prognosis. In this study, we analyzed GATA2mut and co-mutations in 166 Chinese patients with cytogenetically normal AML. This was done through targeted next-generation sequencing of 34 genes associated with myeloid leukemia. GATA2mut was identified in 17 (10%) patients being significantly correlated with co-mutations in CCAAT/enhancer-binding protein alpha (CEBPA) double mutation (P = 0.001). We observed that the N-terminal zinc finger domain (ZF1) was linked to CEBPA mutations, while the C-terminal zinc finger domain (ZF2) was associated with Wilms' tumor 1 (WT1) mutations. It was also noted that patients with GATA2mut had lower platelet counts at diagnosis (P = 0.032). In the entire cohort, GATA2mut had no significant prognostic impact on overall survival (OS) (P = 0.762) and relapse-free survival (RFS) (P = 0.369) compared to patients with GATA2wt. The OS (P = 0.737) and RFS (P = 0.894) of the ZF1 mutation were similar to those of the ZF2 mutation. Most patients with GATA2 mutations were classified in the ELN2022 favorable- and intermediate-risk groups. GATA2mut patients in the favorable-risk group were divided into GATA2High and GATA2Low groups using a median cutoff variant allele frequency (VAF) of 40.13%. GATA2High patients were associated with worse OS (P = 0.031) and RFS (P = 0.021) than GATA2Low patients. In the intermediate-riskgroup, the high median VAF ofGATA2 (≥38.51%) had no significant effect in OS and RFS compared with the low median VAF (<38.51%). This study offers new insights on the prognosis of GATA2mut in the favorable-risk group, where VAF can be used as a guide.

SARS-CoV-2 evolution during prolonged infection in immunocompromised patients.

SARS-CoV-2 is responsible for a global pandemic, driven in part by the emergence of new viral variants. Where do these new variants come from? One model is that long-term viral persistence in infected individuals allows for viral evolution in response to host pressures, resulting in viruses more likely to replicate efficiently in humans. In this study, we characterize replication in several hospitalized and long-term infected individuals, documenting efficient pathways of viral evolution.