Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Development of the selective micropulse individual retinal therapy depends on age and type on the Fitzpatrick scale.

PURPOSE: To develop a selective micropulse individual retinal therapy (SMIRT) based on the age and appearance type of the patient, to derive a formula for calculating power, and evaluate clinical efficacy for the treatment of central serous chorioretinopathy (CSCR). METHODS: 73 patients (aged 30-65 years) with acute CSCR and types 1-4 on the Fitzpatrick scale were divided into 2 groups. In the first group (33 patients), the testing of the micropulse mode (50 µs, 2.4%, 10 ms, 100 µm, 0.4-1.9 W) on the Navilas 577 s laser system defined as selective by computer modeling was performed. A logistic regression function based on probability damage detection (PDD) of the 1584 laser spots from power, age, and type on the Fitzpatrick scale was constructed. PDD is the probability of detecting the laser spots using the autofluorescence method. The second group was divided into 4 subgroups of 10 eyes each. Groups 2.1, 2.2, and 2.3 were treated without preliminary testing. The power for Groups 2.1, 2.2, and 2.3 was obtained with the inverse PDD function, so that PDD was 50%, 70%, and 90%, respectively. Control group 2.4 went without treatment. RESULTS: The transmission and absorption coefficients of laser radiation of the eye depend on the age and the Fitzpatrick scale type. In Groups 2.1-2.3, complete resorption of subretinal fluid was observed 3 months after CSCR treatment in 5 (P < 0.35), 8 (P < 0.023), and 10 eyes (P < 0.0008) out of 10, respectively. CONCLUSION: The developed SMIRT is effective for CSCR treatment with PDD 90%.

Combined PARP and HSP90 inhibition: preclinical and Phase 1 evaluation in patients with advanced solid tumours.

PURPOSE: PARP inhibitor resistance may be overcome by combinatorial strategies with agents that disrupt homologous recombination repair (HRR). Multiple HRR pathway components are HSP90 clients, so that HSP90 inhibition leads to abrogation of HRR and sensitisation to PARP inhibition. We performed in vivo preclinical studies of the HSP90 inhibitor onalespib with olaparib and conducted a Phase 1 combination study. PATIENTS AND METHODS: Tolerability and efficacy studies were performed in patient-derived xenograft(PDX) models of ovarian cancer. Clinical safety, tolerability, steady-state pharmacokinetics and preliminary efficacy of olaparib and onalespib were evaluated using a standard 3 + 3 dose-escalation design. RESULTS: Olaparib/onalespib exhibited anti-tumour activity against BRCA1-mutated PDX models with acquired PARPi resistance and PDX models with RB-pathway alterations(CDKN2A loss and CCNE1 overexpression). Phase 1 evaluation revealed that dose levels up to olaparib 300 mg/onalespib 40 mg and olaparib 200 mg/onalespib 80 mg were safe without dose-limiting toxicities. Coadministration of olaparib and onalespib did not appear to affect the steady-state pharmacokinetics of either agent. There were no objective responses, but disease stabilisation ≥24 weeks was observed in 7/22 (32%) evaluable patients including patients with BRCA-mutated ovarian cancers and acquired PARPi resistance and patients with tumours harbouring RB-pathway alterations. CONCLUSIONS: Combining onalespib and olaparib was feasible and demonstrated preliminary evidence of anti-tumour activity.

Retrosynthetic Analysis of α-Alkenyl-ß-Diketones: Regio- and Stereoselective Two-Step Synthesis of Highly Arylated Representatives from Acetylenes, Ketones, and Acyl Chlorides.

Highly arylated α-alkenyl-ß-diketones are synthesized via a two-step sequence consisting of (i) potassium tert-butoxide/DMSO-catalyzed (E)-stereoselective C-H functionalization of ketones with acetylenes followed by (ii) magnesium bromide etherate/DIPEA-soft enolization of the formed ß,γ-unsaturated ketones and regioselective acylation with acyl chlorides. The method is compatible with a broad range of substrates and shown to be applicable as an intermediate stage in the construction of polyarylated heterocycles.

DNA breaks and chromosome pulverization from errors in mitosis.

The involvement of whole-chromosome aneuploidy in tumorigenesis is the subject of debate, in large part because of the lack of insight into underlying mechanisms. Here we identify a mechanism by which errors in mitotic chromosome segregation generate DNA breaks via the formation of structures called micronuclei. Whole-chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and often extensive fragmentation of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for 'chromothripsis' in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.

Phase II and Biomarker Study of Cabozantinib in Metastatic Triple-Negative Breast Cancer Patients.

Currently, no targeted therapies are available for metastatic triplenegative breast cancer (mTNBC). We evaluated the safety, efficacy, and biomarkers of response to cabozantinib, a multikinase inhibitor, in patients with mTNBC. We conducted a single arm phase II and biomarker study that enrolled patients with measurable mTNBC. Patients received cabozantinib (60 mg daily) on a 3-week cycle and were restaged after 6 weeks and then every 9 weeks. The primary endpoint was objective response rate. Predefined secondary endpoints included progression-free survival (PFS), toxicity, and tissue and blood circulating cell and protein biomarkers. Of 35 patients who initiated protocol therapy, 3 (9% [95% confidence interval (CI): 2, 26]) achieved a partial response (PR). Nine patients achieved stable disease (SD) for at least 15 weeks, and thus the clinical benefit rate (PR+SD) was 34% [95% CI: 19, 52]. Median PFS was 2.0 months [95% CI: 1.3, 3.3]. The most common toxicities were fatigue, diarrhea, mucositis, and palmar-plantar erythrodysesthesia. There were no grade 4 toxicities, but 12 patients (34%) required dose reduction. Two patients had TNBCs with MET amplification. During cabozantinib therapy, there were significant and durable increases in plasma placental growth factor, vascular endothelial growth factor (VEGF), VEGF-D, stromal cell-derived factor 1a, and carbonic anhydrase IX, and circulating CD3 + cells and CD8 + T lymphocytes, and decreases in plasma soluble VEGF receptor 2 and CD14+ monocytes (all p < .05). Higher baseline concentrations of soluble MET (sMET) associated with longer PFS (p = .03). In conclusion, cabozantinib showed encouraging safety and efficacy signals but did not meet the primary endpoint in pretreated mTNBC. Exploratory analyses of circulating biomarkers showed that cabozantinib induces systemic changes consistent with activation of the immune system and antiangiogenic activity, and that sMET should be further evaluated a potential biomarker of response. IMPLICATIONS FOR PRACTICE: Triple-negative breast cancer (TNBC)-a disease with a dearth of effective therapies-often overexpress MET, which is associated with poor clinical outcomes. However, clinical studies of agents targeting MET and VEGF pathways-alone or in combination-have shown disappointing results. This study of cabozantinib (a dual VEGFR2/MET) in metastatic TNBC, while not meeting its prespecified endpoint, showed that treatment is associated with circulating biomarker changes, and is active in a subset of patients. Furthermore, this study demonstrates that cabozantinib therapy induces a systemic increase in cytotoxic lymphocyte populations and a decrease in immunosuppressive myeloid populations. This supports the testing of combinations of cabozantinib with immunotherapy in future studies in breast cancer patients.

Characterization of HPV and host genome interactions in primary head and neck cancers.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution.

FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis.

Multiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Emu-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Emu-XBP-1s elicited elevated serum Ig and skin alterations. With age, Emu-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Emu-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.

Sensitivity to targeted UBA1 inhibition in a myeloid cell line model of VEXAS syndrome.

Somatic UBA1 mutations in hematopoietic cells are a hallmark of Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, which is a late-onset inflammatory disease associated with bone marrow failure and high mortality. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous mutations affecting methionine-41 (M41), leading to the expression of UBA1M41T, UBA1M41V, or UBA1M41L and globally reduced protein polyubiquitination. Here, we used CRISPR-Cas9 to engineer isogenic 32D mouse myeloid cell lines expressing hemizygous Uba1WT or Uba1M41L from the endogenous locus. Consistent with prior analyses of patients with VEXAS syndrome samples, hemizygous Uba1M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform, reduced polyubiquitination, abnormal cytoplasmic vacuoles, and increased production of interleukin-1ß and inflammatory chemokines. Vacuoles in Uba1M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Uba1M41L cells were more sensitive to the UBA1 inhibitor TAK243. TAK243 treatment promoted apoptosis in Uba1M41L cells and led to preferential loss of Uba1M41L cells in competition assays with Uba1WT cells. Knock-in of a TAK243-binding mutation, Uba1A580S, conferred TAK243 resistance. In addition, overexpression of catalytically active UBA1b in Uba1M41L cells restored polyubiquitination and increased TAK243 resistance. Altogether, these data indicate that loss of UBA1b underlies a key biochemical phenotype associated with VEXAS syndrome and renders cells with reduced UBA1 activity vulnerable to targeted UBA1 inhibition. Our Uba1M41L knock-in cell line is a useful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Unveiling the contourite depositional system in the Vema Fracture Zone (Central Atlantic).

A combination of a high sediment input and intense bottom currents often leads to the formation of contourites (sediments deposited or significantly reworked by bottom currents). Both of these components are present in the Vema Fracture Zone valley which is the most important passageway for the distribution of the Antarctic Bottom Water from the West to the North-East of the Atlantic. However, no contourite drifts, moats or contourite channels have been found in this region in more than half a century of research. The prevailing sedimentation paradigm postulates that turbidity currents have predominantly governed sedimentation in this region during the Pleistocene. This work describes the first example of contourite depositional system identified in the Vema Fracture Zone. The discovery was made through detailed high-resolution sub-bottom profiling, as well as numerical modeling and direct measurements of bottom current velocities. Such systems are exceptionally uncommon in fracture zones. This study highlights the importance of further research of contourites along the Vema Fracture Zone based on modern concepts of contourite and mixed depositional systems. The work also emphasizes the need to reevaluate the impact of bottom currents on sedimentation in this region, and particularly in the narrow segments of the fracture zone valley.

Cotargeting a MYC/eIF4A-survival axis improves the efficacy of KRAS inhibitors in lung cancer.

Despite the success of KRAS G12C inhibitors in non-small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to cotarget RAS and mTOR pathways; however, toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identifying the most therapeutically important eukaryotic initiation factor 4F complex-translated (eIF4F-translated) targets. Here, we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.

Development of a Patient-Derived 3D Immuno-Oncology Platform to Potentiate Immunotherapy Responses in Ascites-Derived Circulating Tumor Cells.

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

TRAIL resistance in human neuroblastoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing.

Neuroblastoma is the most common solid extracranial cancer form in childhood with an etiology that is mostly unknown. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising future anticancer drug candidate, highly malignant neuroblastoma has been reported to acquire TRAIL resistance by mechanisms that are poorly understood. Here, we show by western blot analysis, and live cell imaging using anchored FRET sensors, that the resistance to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells depends on an incomplete processing of procaspase-3, generating an immature and catalytically inactive 21 kDa fragment. We have previously shown that the naturally occurring compound curcumin can sensitize SK-N-AS cells to TRAIL. In the present study, we show that curcumin also has a similar effect on human neuroblastoma SHEP1 cells. Furthermore, we show that curcumin and TRAIL co-treatment induces complete maturation and activation of caspase-3 in both cell lines. The mechanisms behind this effect seem to be dependent on protein kinase C (PKC), since inhibition of PKC using bisindolylmaleimide XI, could also sensitize these cells to TRAIL through a similar effect on caspase-3 activation. Moreover, TRAIL co-treatment with bisindolylmaleimide XI or curcumin resulted in down-regulation of X-linked inhibitor of apoptosis protein. In conclusion, our study shows that PKC can be involved in TRAIL resistance in human neuroblastoma cells by preventing caspase-3 maturation.

Transition-metal-free superbase-promoted stereoselective α-vinylation of ketones with arylacetylenes: a general strategy for synthesis of ß,γ-unsaturated ketones.

A wide variety of ß,γ-unsaturated ketones of E configuration have been obtained in good to excellent yields via KO(t)Bu/DMSO promoted α-vinylation of aliphatic, cycloaliphatic, and alkyl aromatic (heteroaromatic) ketones with diverse arylacetylenes.

Determination of Micropulse Modes with Targeted Damage to the Retinal Pigment Epithelium Using Computer Modeling for the Development of Selective Individual Micropulse Retinal Therapy.

PURPOSE: When using a serial laser system for selective impact on the retinal pigment epithelium (RPE), there is a challenge to determine the optimal range of micropulse parameters which result in targeted damage to the RPE. This study proposes a computer model that has identified the optimal parameters to be applied. METHODS: This study was conducted on 18 patients who were diagnosed with acute central serous chorioretinopathy and transparent optical media, aged 35 to 46 years old, and type 2 and 3 on the Fitzpatrick scale. Testing of the micropulse mode was performed on the Navilas 577s laser system; 864 spots were analyzed in total. Considering the probability of damage visualization at different laser power, the computer simulation of tissue heating and protein denaturation was performed to determine the micropulse modes which resulted in selective damage to the RPE. RESULTS: The computer model parameter ΔE = 3.34 × 105 J/mol was determined from fitting the model predictions to the autofluorescence test results. The micropulse modes with a micropulse duration of 50-100 µs, duty cycle 2.4-4.8%, 10 ms-pulse envelope (5 micropulses), and spot diameter of 100 µm have efficiency and selectivity above 67% and correspond to the optimal therapeutic window for targeted RPE damage at a certain power. Increasing the micropulse duration, number of micropulses, and duty cycle leads to a decrease in the selective effect on the RPE and higher damage to adjacent tissues. CONCLUSION: The concepts of efficiency and selectivity have been introduced to quantify the amount of damage caused. The optimal range of micropulse parameters which result in effective and selective damage on the RPE has been determined for the Navilas 577s laser system. The proposed method can be used for any other serial laser system. A comparison of the different micropulse modes, as well as the CW modes, has been performed.

Genome sequence data and properties of Bifidobacterium bifidum strain ICIS-504 isolated from multispecies bifidobacterial community.

This report presents the data on the draft genome sequence of Bifidobacterium bifidum strain ICIS-504. The strain, isolated from the intestine of a 41-year-old healthy woman is a member of community consist of four strains of three bifidobacterial species: B. longum, B. bifidum and B. breve. Annotation of the genome sequence revealed as high similarity with deposited strains as the unique duplication in the functional region of the only AmiR-family response regulator gene. The draft genome sequence data of B. bifidum strain ICIS-504 is available under the accession nos. JAJJPE000000000.1, PRJNA776132 and SAMN22746550 for NCBI Genome, Bioproject and Biosample databases, respectively.

Semen microbiota and cytokines of healthy and infertile men.

In this study, we determined the levels of cytokine secretory inhibitors and the microbiota biofilms of semen from healthy and infertile subjects. A total of 118 clinical bacterial isolates were isolated and tested. Cytokine secretory inhibitors were determined based on the difference in cytokine content between the control and experimental samples of cell-free supernatants of isolated microorganisms. Biofilm formation was studied by determining the adhesion of microorganisms to the surface of a 96-well sterile plate and expressed as the optical density at 630 nm (OD630). Cell-free supernatants of Staphylococcus contained higher levels of secretory inhibitor of cytokines in conditionally healthy than in infertile patients. In contrast, in infertile men, the ability to reduce cytokine levels was more characteristic of Enterococcus and Corynebacterium. Seminal Staphylococcus, Corynebacterium, and Enterococcus isolated from infertile subjects showed a greater ability to form biofilms than the same bacteria isolated from healthy men. Further research is needed on this topic, since it is necessary to determine the relationships between decreased secretory inhibitors of cytokines, production of biofilms by bacteria in semen, and infertility. It is likely that the ability of microorganisms to change the concentration of cytokines and increase the level of biofilm formation in semen may be associated with minimal impairments of fertilizing ability, which are not detected using other methods.

EGFR Inhibition Enhances the Cellular Uptake and Antitumor-Activity of the HER3 Antibody-Drug Conjugate HER3-DXd.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) are the standard-of-care treatment for EGFR-mutant non-small cell lung cancers (NSCLC). However, most patients develop acquired drug resistance to EGFR TKIs. HER3 is a unique pseudokinase member of the ERBB family that functions by dimerizing with other ERBB family members (EGFR and HER2) and is frequently overexpressed in EGFR-mutant NSCLC. Although EGFR TKI resistance mechanisms do not lead to alterations in HER3, we hypothesized that targeting HER3 might improve efficacy of EGFR TKI. HER3-DXd is an antibody-drug conjugate (ADC) comprised of HER3-targeting antibody linked to a topoisomerase I inhibitor currently in clinical development. In this study, we evaluated the efficacy of HER3-DXd across a series of EGFR inhibitor-resistant, patient-derived xenografts and observed it to be broadly effective in HER3-expressing cancers. We further developed a preclinical strategy to enhance the efficacy of HER3-DXd through osimertinib pretreatment, which increased membrane expression of HER3 and led to enhanced internalization and efficacy of HER3-DXd. The combination of osimertinib and HER3-DXd may be an effective treatment approach and should be evaluated in future clinical trials in EGFR-mutant NSCLC patients. SIGNIFICANCE: EGFR inhibition leads to increased HER3 membrane expression and promotes HER3-DXd ADC internalization and efficacy, supporting the clinical development of the EGFR inhibitor/HER3-DXd combination in EGFR-mutant lung cancer.See related commentary by Lim et al., p. 18.