Exploring the PET in vivo generator 134Ce as a theranostic match for 225Ac.

Purpose: The radionuclide pair cerium-134/lanthanum-134 (134Ce/134La) was recently proposed as a suitable diagnostic counterpart for the therapeutic alpha-emitter actinium-225 (225Ac). The unique properties of 134Ce offer perspectives for developing innovative in vivo investigations not possible with 225Ac. In this work, 225Ac- and 134Ce-labeled tracers were directly compared using internalizing and slow-internalizing cancer models to evaluate their in vivo comparability, progeny meandering, and potential as a matched theranostic pair for clinical translation. Despite being an excellent chemical match, 134Ce/134La has limitations to the setting of quantitative positron emission tomography imaging. Methods: The precursor PSMA-617 and a macropa-based tetrazine-conjugate (mcp-PEG8-Tz) were radiolabelled with 225Ac or 134Ce and compared in vitro and in vivo using standard (radio)chemical methods. Employing biodistribution studies and positron emission tomography (PET) imaging in athymic nude mice, the radiolabelled PSMA-617 tracers were evaluated in a PC3/PIP (PC3 engineered to express a high level of prostate-specific membrane antigen) prostate cancer mouse model. The 225Ac and 134Ce-labeled mcp-PEG8-Tz were investigated in a BxPC-3 pancreatic tumour model harnessing the pretargeting strategy based on a trans-cyclooctene-modified 5B1 monoclonal antibody. Results: In vitro and in vivo studies with both 225Ac and 134Ce-labelled tracers led to comparable results, confirming the matching pharmacokinetics of this theranostic pair. However, PET imaging of the 134Ce-labelled precursors indicated that quantification is highly dependent on tracer internalization due to the redistribution of 134Ce's PET-compatible daughter 134La. Consequently, radiotracers based on internalizing vectors like PSMA-617 are suited for this theranostic pair, while slow-internalizing 225Ac-labelled tracers are not quantitatively represented by 134Ce PET imaging. Conclusion: When employing slow-internalizing vectors, 134Ce might not be an ideal match for 225Ac due to the underestimation of tumour uptake caused by the in vivo redistribution of 134La. However, this same characteristic makes it possible to estimate the redistribution of 225Ac's progeny noninvasively. In future studies, this unique PET in vivo generator will further be harnessed to study tracer internalization, trafficking of receptors, and the progression of the tumour microenvironment.

Phase I/II Study of Combined BCL-xL and MEK Inhibition with Navitoclax and Trametinib in KRAS or NRAS Mutant Advanced Solid Tumors.

PURPOSE: MEK inhibitors (MEKi) lack monotherapy efficacy in most RAS-mutant cancers. BCL-xL is an anti-apoptotic protein identified by a synthetic lethal shRNA screen as a key suppressor of apoptotic response to MEKi. PATIENTS AND METHODS: We conducted a dose escalation study (NCT02079740) of the BCL-xL inhibitor navitoclax and MEKi trametinib in patients with RAS-mutant tumors with expansion cohorts for: pancreatic, gynecologic (GYN), non-small cell lung cancer (NSCLC), and other cancers harboring KRAS/NRAS mutations. Paired pretreatment and day 15 tumor biopsies and serial cell-free (cf)DNA were analyzed. RESULTS: A total of 91 patients initiated treatment, with 38 in dose escalation. Fifty-eight percent had ≥3 prior therapies. A total of 15 patients (17%) had colorectal cancer, 19 (11%) pancreatic, 15 (17%) NSCLC, and 32 (35%) GYN cancers. The recommended phase II dose (RP2D) was established as trametinib 2 mg daily days 1 to 14 and navitoclax 250 mg daily days 1 to 28 of each cycle. Most common adverse events included diarrhea, thrombocytopenia, increased AST/ALT, and acneiform rash. At RP2D, 8 of 49 (16%) evaluable patients achieved partial response (PR). Disease-specific differences in efficacy were noted. In patients with GYN at the RP2D, 7 of 21 (33%) achieved a PR and median duration of response 8.2 months. No PRs occurred in patients with colorectal cancer, NSCLC, or pancreatic cancer. MAPK pathway inhibition was observed in on-treatment tumor biopsies. Reductions in KRAS/NRAS mutation levels in cfDNA correlated with clinical benefit. CONCLUSIONS: Navitoclax in combination with trametinib was tolerable. Durable clinical responses were observed in patients with RAS-mutant GYN cancers, warranting further evaluation in this population.

Development of immunoliposomes containing cytotoxic gold payloads against HER2-positive breast cancers.

Overexpression of the human epidermal growth factor receptor 2 (HER2) is found in 20-30% of breast cancer tumors (HER2-positive breast cancers) and is associated with more aggressive onset of disease, higher recurrence rate and increased mortality. Monoclonal antibodies (mAb) like trastuzumab and pertuzumab in combination with chemotherapeutics, and trastuzumab-based antibody drug conjugates (ADCs) are used in the clinic to treat these cancers. An alternative targeted strategy (not yet in clinical use) is the encapsulation of chemotherapeutic drugs in immunoliposomes. Such systems may not only facilitate targeted delivery to the tumor and improve intracellular penetration, but also override some of the resistance developed by tumors in response to cytotoxic loads. As a supplement to classical chemotherapeutics (based on organic compounds and conventional platinum-based derivatives), gold compounds are emerging as potential anticancer agents due to their high cytotoxicity and capacity for immunogenic cell death. Here, we describe the development of immunoliposomes functionalized with trastuzumab and pertuzumab; containing simple gold(i) neutral compounds ([AuCl(PR3)] (PR3 = PPh3 (1), PEt3 (2))) generated by the thin-film method to afford Lipo-1-Lipo-2. Trastuzumab and pertuzumab were engrafted onto these liposomes to generate gold-based immunoliposomes (Immunolipo-Tras-1, Immunolipo-Tras-2, Immunolipo-Per-1, Immunolipo-Per-2). We have characterized all liposomal formulations and demonstrated that the immunoliposomes (190 nm) are stable, have high binding affinity for HER2, and display selective cytotoxicity towards HER2-positive breast cancer cell lines. Trastuzumab-based immunoliposomes of a smaller size (100 nm) - encapsulating [AuCl(PEt3)] (2) - have been generated by an extrusion homogenization method. These optimized immunoliposomes (Opt-Immunolipo-Tras-2) have a trastuzumab engraftment efficiency, encapsulation efficiency for 2, and affinity for HER-2 similar to the immunoliposomes obtained by sonication (Immunolipo-Tras-2). While the amount of Au encapsulated is slightly lower, they display almost identical cytotoxicity and selectivity profiles. Moreover, the fluorescently-labeled phosphane drug [AuCl(PPh2-BODIPY)] (3) was encapsulated in both larger (Immunolipo-Tras-3) and smaller (Opt-Immunolipo-Tras-3) immunoliposomes and used to visualize the intracellular localization of the payload. Fluorescent imaging studies found that Opt-Immunolipo-Tras-3 accumulates in the cells more than 3 and that the unencapsulated payload accumulates primarily in lysosomes, while targeted liposomal 3 localizes in mitochondria and ER, hinting at different possibilities for modes of action.

Correction to "Shifting the Antibody-Drug Conjugate Paradigm: A Trastuzumab-Gold-Based Conjugate Demonstrates High Efficacy against Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer Mouse Model".

[This corrects the article DOI: 10.1021/acsptsci.3c00270.].

212Pb-Pretargeted Theranostics for Pancreatic Cancer.

Although pancreatic ductal adenocarcinoma (PDAC) is associated with limited treatment options and poor patient outcomes, targeted α-particle therapy (TAT) represents a promising development in the field. TAT shows potential in treating metastatic cancers, including those that have become resistant to conventional treatments. Among the most auspicious radionuclides stands the in vivo α-generator 212Pb. Combined with the imaging-compatible radionuclide 203Pb, this theranostic match is a promising modality rapidly translating into the clinic. Methods: Using the pretargeting approach between a radiolabeled 1,2,4,5-tetrazine (Tz) tracer and a trans-cyclooctene (TCO) modified antibody, imaging and therapy with radiolead were performed on a PDAC tumor xenograft mouse model. For therapy, 3 cohorts received a single administration of 1.1, 2.2, or 3.7 MBq of the pretargeting agent, [212Pb]Pb-DO3A-PEG7-Tz, whereby administered activity levels were guided by dosimetric analysis. Results: The treated mice were holistically evaluated; minimal-to-mild renal tubular necrosis was observed. At the same time, median survival doubled for the highest-dose cohort (10.7 wk) compared with the control cohort (5.1 wk). Conclusion: This foundational study demonstrated the feasibility and safety of pretargeted TAT with 212Pb in PDAC while considering dose limitations and potential adverse effects.

Landscape of Clinical Resistance Mechanisms to FGFR Inhibitors in FGFR2-Altered Cholangiocarcinoma.

PURPOSE: FGFR inhibitors are effective in FGFR2-altered cholangiocarcinoma, leading to approval of reversible FGFR inhibitors, pemigatinib and infigratinib, and an irreversible inhibitor, futibatinib. However, acquired resistance develops, limiting clinical benefit. Some mechanisms of resistance have been reported, including secondary FGFR2 kinase domain mutations. Here, we sought to establish the landscape of acquired resistance to FGFR inhibition and to validate findings in model systems. EXPERIMENTAL DESIGN: We examined the spectrum of acquired resistance mechanisms detected in circulating tumor DNA or tumor tissue upon disease progression following FGFR inhibitor therapy in 82 FGFR2-altered cholangiocarcinoma patients from 12 published reports. Functional studies of candidate resistance alterations were performed. RESULTS: Overall, 49 of 82 patients (60%) had one or more detectable secondary FGFR2 kinase domain mutations upon acquired resistance. N550 molecular brake and V565 gatekeeper mutations were most common, representing 63% and 47% of all FGFR2 kinase domain mutations, respectively. Functional studies showed different inhibitors displayed unique activity profiles against FGFR2 mutations. Interestingly, disruption of the cysteine residue covalently bound by futibatinib (FGFR2 C492) was rare, observed in 1 of 42 patients treated with this drug. FGFR2 C492 mutations were insensitive to inhibition by futibatinib but showed reduced signaling activity, potentially explaining their low frequency. CONCLUSIONS: These data support secondary FGFR2 kinase domain mutations as the primary mode of acquired resistance to FGFR inhibitors, most commonly N550 and V565 mutations. Thus, development of combination strategies and next-generation FGFR inhibitors targeting the full spectrum of FGFR2 resistance mutations will be critical.

Shifting the Antibody-Drug Conjugate Paradigm: A Trastuzumab-Gold-Based Conjugate Demonstrates High Efficacy against Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer Mouse Model.

Antibody-drug conjugates (ADCs) combine the selectivity of monoclonal antibodies (mAbs) with the efficacy of chemotherapeutics to target cancers without toxicity to normal tissue. Clinically, most chemotherapeutic ADCs are based on complex organic molecules, while the conjugation of metallodrugs to mAbs has been overlooked, despite the resurgent interest in metal-based drugs as cancer chemotherapeutics. In 2019, we described the first gold ADCs containing gold-triphenylphosphane fragments as a proof of concept. The ADCs (based on the antibody trastuzumab) were selective and highly active against HER2-positive breast cancer cells. In this study, we developed site-specific ADCs (Thio-1b and Thio-2b) using the cysteine-engineered trastuzumab derivative THIOMAB antibody technology with gold(I)-containing phosphanes and a maleimide-based linker amenable to bioconjugation (1b and 2b). In addition, we developed lysine-directed ADCs with gold payloads based on phosphanes and N-heterocyclic carbenes featuring an activated ester moiety (2c and 5c) with trastuzumab (Tras-2c and Tras-5c) and another anti-HER2 antibody, pertuzumab (Per-2c and Per-5c). Both sets of ADCs demonstrated significant anticancer potency in vitro assays. Based on these results, one ADC (Tras-2c), containing the [Au(PEt3)] fragment present in FDA-approved auranofin, was selected for an in vivo antitumor efficacy study. Immunocompromised mice xenografted with the HER2-positive human cancer cell line SKBR-3 exhibited almost complete tumor reduction and low toxicity with intravenous administration of Tras-2c. With this highly selective targeting system, we demonstrated that a subnanomolar cytotoxicity profile in cells is not required for an impressive antitumor effect in a mouse xenograft model.

89Zr-DFO-Isatuximab for CD38-Targeted ImmunoPET Imaging of Multiple Myeloma and Lymphomas.

Multiple myeloma (MM) is the second most prevalent hematological malignancy. It remains incurable despite the availability of novel therapeutic approaches, marking an urgent need for new agents for noninvasive targeted imaging of MM lesions. CD38 has proven to be an excellent biomarker due to its high expression in aberrant lymphoid and myeloid cells relative to normal cell populations. Using isatuximab (Sanofi), the latest FDA-approved CD38-targeting antibody, we have developed Zirconium-89(89Zr)-labeled isatuximab as a novel immunoPET tracer for the in vivo delineation of MM and evaluated the extension of its applicability to lymphomas. In vitro studies validated the high binding affinity and specificity of 89Zr-DFO-isatuximab for CD38. PET imaging demonstrated the high performance of 89Zr-DFO-isatuximab as a targeted imaging agent to delineate tumor burden in disseminated models of MM and Burkitt's lymphoma. Ex vivo biodistribution studies confirmed that high accumulations of the tracer in bone marrow and bone skeleton correspond to specific disease lesions as they are reduced to background in blocking and healthy controls. This work demonstrates the promise of 89Zr-DFO-isatuximab as an immunoPET tracer for CD38-targeted imaging of MM and certain lymphomas. More importantly, its potential as an alternative to 89Zr-DFO-daratumumab holds great clinical relevance.

Combined PD-1, BRAF and MEK inhibition in BRAFV600E colorectal cancer: a phase 2 trial.

While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and lack durability. Preclinical data suggest that BRAF/MAPK pathway inhibition may augment the tumor immune response. We performed a proof-of-concept single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC. The primary end point was overall response rate, and the secondary end points were progression-free survival, disease control rate, duration of response and overall survival. The study met its primary end point with a confirmed response rate (24.3% in all patients; 25% in microsatellite stable patients) and durability that were favorable relative to historical controls of BRAF-targeted combinations alone. Single-cell RNA sequencing of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed greater induction of tumor cell-intrinsic immune programs and more complete MAPK inhibition in patients with better clinical outcome. Immune program induction in matched patient-derived organoids correlated with the degree of MAPK inhibition. These data suggest a potential tumor cell-intrinsic mechanism of cooperativity between MAPK inhibition and immune response, warranting further clinical evaluation of optimized targeted and immune combinations in CRC. ClinicalTrials.gov registration: NCT03668431.

Comparison of Two-Bag Versus Three-Bag N-Acetylcysteine Regimens for Pediatric Acetaminophen Toxicity.

BACKGROUND: Acetaminophen overdose is a leading cause of liver failure, and a leading cause of pediatric poisoning requiring hospital admission. The antidote, N-acetylcysteine (NAC), is traditionally administered as a three-bag intravenous infusion. Despite its efficacy, NAC is associated with high incidence of nonallergic anaphylactoid reactions (NAARs). Adult evidence demonstrates that alternative dosing regimens decrease NAARs and medication errors (MEs). OBJECTIVES: To compare NAARs and MEs associated with two- versus three-bag NAC for acetaminophen overdose in a pediatric population. METHODS: This is a retrospective observational cohort study comparing pediatric patients who received three- versus two-bag NAC for acetaminophen toxicity. The primary outcome was incidence of NAARs. Secondary outcomes were rates of MEs and relevant hospital outcomes (length of stay [LOS], intensive care unit (ICU) admission, liver transplant, death). RESULTS: Two hundred forty-three patients met inclusion criteria (median age of 15 years): 150 (62%) three-bag NAC and 93 (38%) two-bag NAC. There was no difference in overall NAARs (p = 0.54). Fewer cutaneous NAARs were observed in the two-bag group, three-bag: 15 (10%), two-bag: 2 (2%), p = 0.02. MEs were significantly decreased with the two-bag regimen, three-bag: 59 (39%), two-bag: 21 (23%), p = 0.01. No statistical differences were observed in LOS, ICU admissions, transplant, or death. CONCLUSION AND RELEVANCE: A significant decrease in cutaneous NAARs and MEs was observed in pediatric patients by combining the first two bags of the traditional three-bag NAC regimen. In pediatric populations, a two-bag NAC regimen for acetaminophen overdose may improve medication tolerance and safety.

Incidence and management of esophageal cancer recurrence to regional lymph nodes after curative esophagectomy.

Up to 50% of patients treated with curative esophagectomy for esophageal cancer will develop recurrence, contributing to the dismal survival associated with this disease. Regional recurrence may represent disease that is not yet widely metastatic and may therefore be amenable to more-aggressive treatment. We sought to assess all patients treated with curative esophagectomy for esophageal cancer who developed regional recurrence. We retrospectively identified all patients who underwent esophagectomy for esophageal adenocarcinoma and esophageal squamous cell carcinoma at a single institution from January 2000 to August 2019. In total, 1626 patients were included in the study cohort. As of June 2022, 595 patients had disease recurrence, which was distant or systemic in 435 patients (27%), regional in 125 (7.7%) and local in 35 (2.2%). On multivariable analysis, neoadjuvant chemoradiation with a total radiation dose <45 Gy (hazard ratio [HR], 3.5 [95% CI, 1.7-7.3]; P = .001), pathologic node-positive disease (HR, 1.9 [95% CI, 1.3-3.0]; P = .003) and lymphovascular invasion (HR, 1.6 [95% CI, 1.0-2.5]; P = .049) were predictors of isolated nodal recurrence, whereas increasing age (HR, 0.97 [95% CI, 0.96-0.99]; P = .001) and increasing number of excised lymph nodes (HR, 0.98 [95% CI, 0.95-1.00]; P = .021) were independently associated with decreased risk of regional recurrence. Patients treated with a combination of local and systemic therapies had better survival outcomes than patients treated with systemic therapy alone (P < .001). In patients with recurrence of esophageal cancer limited to regional lymph nodes, salvage treatment may be possible. Higher radiation doses and more-extensive lymphadenectomy may reduce the risk of regional recurrence.

Polyethylene Glycol 3350 (PEG 3350) as a Practical Vehicle for Rapid Reconstitution of PARPi-FL Formulations for Clinical Use.

PARPi-FL is a molecularly specific fluorescent agent that targets poly ADP-ribose polymerase 1, a DNA repair enzyme overexpressed in the nuclei of tumor cells. This imaging agent is being investigated in a clinical trial (NCT03085147) for the detection of oral cancer. The PARPi-FL mouthwash formulation currently being used in the phase I/II clinical trial comprises 1,000 nM of PARPi-FL dissolved first in 4.5 ml of polyethylene glycol (PEG) 300 and then in 9.5 ml of water. This formulation requires a 2-step process that can be cumbersome for routine clinical use. To minimize errors and simplify the formulation process, we have developed a new one-step formulation, which requires only the direct addition of water into a vial containing a mixture of the PARPi-FL and PEG 3350, which is also a powder. In a series of analytical and preclinical studies, we demonstrate that the new formulation of PARPi-FL is stable over 365 days, sustains its characteristics, and performs similar to the previous formulation. Moving forward, the new formulation of the PARPi-FL will be used for patients accrued in the phase II clinical trial.

Cell-free human papillomavirus DNA kinetics after surgery for human papillomavirus-associated oropharyngeal cancer.

BACKGROUND: New ultrasensitive methods for detecting residual disease after surgery are needed in human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC). METHODS: To determine whether the clearance kinetics of circulating tumor human papillomavirus DNA (ctHPVDNA) is associated with postoperative disease status, a prospective observational study was conducted in 33 patients with HPV+OPSCC undergoing surgery. Blood was collected before surgery, postoperative days 1 (POD 1), 7, and 30 and with follow-up. A subcohort of 12 patients underwent frequent blood collections in the first 24 hours after surgery to define early clearance kinetics. Plasma was run on custom droplet digital polymerase chain reaction (ddPCR) assays for HPV genotypes 16, 18, 33, 35, and 45. RESULTS: In patients without pathologic risk factors for recurrence who were observed after surgery, ctHPVDNA rapidly decreased to <1 copy/mL by POD 1 (n = 8/8). In patients with risk factors for macroscopic residual disease, ctHPVDNA was markedly elevated on POD 1 (>350 copies/mL) and remained elevated until adjuvant treatment (n = 3/3). Patients with intermediate POD 1 ctHPVDNA levels (1.2-58.4 copies/mL) all possessed pathologic risk factors for microscopic residual disease (n = 9/9). POD 1 ctHPVDNA levels were higher in patients with known adverse pathologic risk factors such as extranodal extension >1 mm (P = .0481) and with increasing lymph nodes involved (P = .0453) and were further associated with adjuvant treatment received (P = .0076). One of 33 patients had a recurrence that was detected by ctHPVDNA 2 months earlier than clinical detection. CONCLUSIONS: POD 1 ctHPVDNA levels are associated with the risk of residual disease in patients with HPV+OPSCC undergoing curative intent surgery and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future. LAY SUMMARY: Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC) is increasing at epidemic proportions and is commonly treated with surgery. This report describes results from a study examining the clearance kinetics of circulating tumor HPV DNA (circulating tumor human papillomavirus DNA [ctHPVDNA]) following surgical treatment of HPV+OPSCC. We found that ctHPVDNA levels 1 day after surgery are associated with the risk of residual disease in patients with HPV+OPSCC and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future. These findings are the first to demonstrate the potential utility of ctHPVDNA in patients with HPV+OPSCC undergoing surgery.

Cell-Free HPV DNA Provides an Accurate and Rapid Diagnosis of HPV-Associated Head and Neck Cancer.

PURPOSE: HPV-associated head and neck squamous cell carcinoma (HPV+HNSCC) is the most common HPV-associated malignancy in the United States and continues to increase in incidence. Current diagnostic approaches for HPV+HNSCC rely on tissue biopsy followed by histomorphologic assessment and detection of HPV indirectly by p16 IHC. Such approaches are invasive and have variable sensitivity. EXPERIMENTAL DESIGN: We conducted a prospective observational study in 140 subjects (70 cases and 70 controls) to test the hypothesis that a noninvasive diagnostic approach for HPV+HNSCC would have improved diagnostic accuracy, lower cost, and shorter diagnostic interval compared with standard approaches. Blood was collected, processed for circulating tumor HPV DNA (ctHPVDNA), and analyzed with custom ddPCR assays for HPV genotypes 16, 18, 33, 35, and 45. Diagnostic performance, cost, and diagnostic interval were calculated for standard clinical workup and compared with a noninvasive approach using ctHPVDNA combined with cross-sectional imaging and physical examination findings. RESULTS: Sensitivity and specificity of ctHPVDNA for detecting HPV+HNSCC were 98.4% and 98.6%, respectively. Sensitivity and specificity of a composite noninvasive diagnostic using ctHPVDNA and imaging/physical examination were 95.1% and 98.6%, respectively. Diagnostic accuracy of this noninvasive approach was significantly higher than standard of care (Youden index 0.937 vs. 0.707, P = 0.0006). Costs of noninvasive diagnostic were 36% to 38% less than standard clinical workup and the median diagnostic interval was 26 days less. CONCLUSIONS: A noninvasive diagnostic approach for HPV+HNSCC demonstrated improved accuracy, reduced cost, and a shorter time to diagnosis compared with standard clinical workup and could be a viable alternative in the future.

Clinical Acquired Resistance to KRASG12C Inhibition through a Novel KRAS Switch-II Pocket Mutation and Polyclonal Alterations Converging on RAS-MAPK Reactivation.

Mutant-selective KRASG12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRAS G12C-mutant cancers, including non-small cell lung cancer (NSCLC). However, mechanisms underlying clinical acquired resistance to KRASG12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a patient with KRAS G12C NSCLC who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS-MAPK signaling. Notably, a novel KRAS Y96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein-drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRAS G12C cancer models. Interestingly, a novel, functionally distinct tricomplex KRASG12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRASG12C/Y96D and could overcome resistance. SIGNIFICANCE: In one of the first reports of clinical acquired resistance to KRASG12C inhibitors, our data suggest polyclonal RAS-MAPK reactivation as a central resistance mechanism. We also identify a novel KRAS switch-II pocket mutation that impairs binding and drives resistance to inactive-state inhibitors but is surmountable by a functionally distinct KRASG12C inhibitor.See related commentary by Pinnelli and Trusolino, p. 1874.This article is highlighted in the In This Issue feature, p. 1861.