Investigational Agents Research Articles

Abstract 4560: Assessing cancer drug combination efficacy across 900+ PRISM cell lines in a multiplexed screening assay

Abstract Combination therapies are crucial in cancer care, yet identifying which combination will benefit a specific patient is challenging. The vast array of clinical, investigational, and tool anticancer agents and diverse cancer contexts makes investigating many drug combinations over a large number of cell lines infeasible. Here, we describe a multiplexed screening approach using the PRISM assay, where drug combinations are screened on >900 DNA-barcoded cancer cell lines. We analyze cell viability data in drug combinations and single agents using established metrics to describe the combined effects such as additivity, synergy, and antagonism. Quantifying and classifying combined effects will help prioritize specific combinations for follow-up investigations and provide biological and mechanistic insights, thus enabling rationally designed combination therapies. We illustrate our methodology through data from three drug combinations that exemplify synergy and antagonism. In the first combination, temozolomide+O6-benzylguanine (alkylating agent + MGMT inhibitor), we found that cell lines expressing MGMT were sensitized to temozolomide in the presence of O6-benzylguanine, thus illustrating synergy. In the second combination, we screened two anti-apoptosis compounds A-1331852+AZD5991 (BCL-xL + MCL1 inhibitor) and found that BCL-xL and MCL1 inhibition were also synergistic. In the third combination, ML210+ferrostatin-1 (GPX4 inhibitor inducing ferroptosis + ferroptosis inhibitor), we found that ferrostatin-1 antagonized the effects of ML210. Our analysis also reveals the importance of appropriate dose selection and analytical metrics for reliably measuring combined effects. In conclusion, the PRISM platform's multiplexed cell line screening is a robust method for characterizing rationally designed drug combinations, offering a systematic approach to enhance the precision of combination therapy development in cancer care. Citation Format: Ashish Bino George, Shiker Nair, Mustafa Kocak, Ellen Nguyen, Alvin Kalathungal, Anthony Fazio, Cole Ponsi, Aydin Golabi, Melissa A. Ronan, Matthew G. Rees, Jennifer A. Roth. Assessing cancer drug combination efficacy across 900+ PRISM cell lines in a multiplexed screening assay [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 4560.

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 1217: Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma

Abstract Introduction: Mutations in the small GTPase NRAS are the second most common oncogenic driver in cutaneous melanoma, being found in ~25% of cases. Targeted therapy strategies are currently lacking for NRAS-mutant melanoma. Here, we characterized the anti-tumor activity and immune effects of RMC-7977, a RASMULTI(ON) preclinical tool compound, representative of the investigational agent RMC-6236, in preclinical mouse and human models of NRAS-mutant melanoma. Methods: The in vitro potency of RMC-7977 was determined using mouse and human NRAS-mutant melanoma cell lines. Recovery of signaling was quantified by Western Blot and receptor tyrosine kinase (RTK) arrays. In vivo anti-tumor activity of RMC-7977 was determined in two syngeneic mouse melanoma models (SW1 NRASG13D and NRAS#5 NRASQ61R), and tumor immune infiltrate quantified using multiplexed IHC and flow cytometry. Mechanisms of resistance were defined using RNA-seq. Results: RMC-7977 had good activity in vitro against human cancer cell lines and in mouse models of NRAS-mutant melanoma. Time-course studies demonstrated RMC-7977 rapidly inhibited phospho-ERK signaling, which then began to recover after 8 hr of treatment. There was also evidence of adaptive receptor tyrosine kinase signaling by 48 hr. In syngeneic mouse models of NRAS-mutant melanoma, RMC-7977 led to a rapid inhibition of tumor growth, increased expression of tumor MHC class I and PD-L1 and an increase in CD4+ and CD8+ T cell infiltrate into tumors. Therapeutic escape was associated with decreased CD4+ and CD8+ T cell infiltration and increased numbers of immunosuppressive MDSCs. Cells collected from tumors resistant to RMC-7977 in vivo did not show any shift in sensitivity to the inhibitor when re-challenged in vitro, suggesting that the resistance observed in vivo resulted from mechanisms specific to the tumor microenvironment, for example adaptive RTK signaling or immune escape. RNA-seq and IHC analysis suggested that resistance was associated with decreased MHC expression and antigen presentation, as well as reduced co-stimulation of T cells. Combination studies of RMC-7977 with anti-PD-1 are ongoing to identify potential synergistic interactions in these preclinical models. Conclusion: RMC-7977 has anti-tumor activity in syngeneic mouse models of NRAS-mutant melanoma, associated with a robust immune response. Citation Format: Larissa A. Carvalho, Manali S. Phadke, Keiran S. M. Smalley. Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma [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 1217.

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 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 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.

Primary biliary cholangitis-response criteria of first-line treatment and perspectives of second-line therapy

Primary biliary cholangitis (PBC) is achronic cholestatic liver disease that can progress to liver cirrhosis if left untreated. Early diagnosis, initiation of therapy and, if necessary, adjustment of treatment are essential to prevent disease progression. The timing and thresholds for assessing adequate treatment response are inconsistently defined in the literature and can pose achallenge in clinical practice. In addition to providing aconcise overview of the guideline-based diagnostic work-up and first-line therapy, this study offers practical guidance for the evaluation of treatment response and options for second-line treatment in PBC. This article is based on the current European Association for the Study of the Liver (EASL) clinical practice guidelines for the management of PBC from 2017 as well as aliterature review of studies from 2017 to 2023, focusing on defining treatment response, assessing disease progression risk, and the approved and investigational agents for second-line therapy. There are varying definitions for asufficient response to ursodeoxycholic acid (UDCA). Therapeutic goals are tailored to the individual risk of disease progression. The lowest risk appears to be associated with normalization of alkaline phosphatase (AP) and serum bilirubin below 0.6 the upper limit of normal. Established second-line therapies include obeticholic acid and bezafibrate (off-label use), while other peroxisome proliferator-activated receptor (PPAR) agonists and combination therapies are under clinical investigation. Early evaluation of treatment response to UDCA is mandatory. In the case of insufficient treatment response, second-line therapy should be initiated according to the individual's risk profile.

Abstract PR-002: Activation of the integrated stress response by NXP800, an orally available, clinical-stage, investigational agent in ARID1A-mutated, platinum resistant ovarian cancer

Abstract Using innovative phenotypic screening, targeting the Heat Shock Factor 1 (HSF1) pathway, followed by multiparameter medicinal chemistry optimization, we discovered NXP800, an orally active, potent inhibitor of cell proliferation. Evaluation in a mini-panel of human cancer cell lines and tumor xenografts revealed high sensitivity in ARIDIA-deficient human ovarian cancer models, confirmed in the large Sanger panel and isogenic systems. By RNAseq we identified overlapping gene expression changes in human cancer cell lines exposed to NXP800, including expected changes in HSF1-regulated genes plus alterations in ATF4-regulated gene expression associated with activation of the integrated stress response (ISR). This did not indicate a global stress response to NXP800 as we saw no activation of the unfolded protein response. Consistent with activation of the ISR, NXP800 induced phosphorylation of EIF2A and increased expression of downstream ISR markers/effectors ATF4, CHAC1 and CHOP both in human ovarian cells in vitro and corresponding tumor xenograft models in vivo. Using an siRNA approach, we found that blocking the induction of ATF4 reduced the response of sensitive, ARID1A mutant SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. Phosphorylation of EIF2A is tightly regulated by four stress-controlled kinases, GCN2, HRI, PKR and PERK. Using either systematic siRNA knockdown or inhibition by two small-molecule tool compounds from different chemotypes, we discovered that GCN2 alone was required for ISR activation by NXP800. Also, inactivation of GCN2 markedly reduced the antiproliferative activity of NXP800. Global phospho-proteome analysis demonstrated defined changes in response to NXP800 which were reversed on co-treatment with a GCN2 inhibitor. Furthermore, ISR induction inhibited HSF1 activation, confirming the mechanistic link between ISR activation and inhibition of HSF1-mediated transcription. In summary, we discovered the mechanistically novel drug NXP800 which acts on cancer cells to stimulate GCN2 and thereby activate the ISR pathway, leading to inhibition of cap-dependent protein translation. NXP800 shows highly promising activity in human ovarian cancer, including tumor regression of ARID1A-deficient ovarian cancer xenografts. Studies are currently underway to determine precisely how NXP800 stimulates GCN2 activity and the role of ARID1A deficiency. With Nuvectis Pharma, the Phase 1a dose escalation study is completing and the multicentre Phase 1b expansion cohort study in platinum-resistant ARID1A-mutated ovarian cancer is now initiated (NCT05226507) in collaboration with the GOG Foundation and the European Network of Gynecological Oncological Trial Group (ENGOT). FDA has issued a Fast Track designation to NXP800 in this setting. Citation Format: Marissa V. Powers, Swee Y. Swap, Robert te Poele, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Suzenne Eccles, Florence Raynaud, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by NXP800, an orally available, clinical-stage, investigational agent in ARID1A-mutated, platinum resistant ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-002.

Next-Generation HER2-Targeted Antibody-Drug Conjugates in Breast Cancer.

Human epidermal growth factor receptor 2 (HER2) tyrosine kinase is overexpressed in 20% of breast cancers and associated with a less favorable prognosis compared to HER2-negative disease. Patients have traditionally been treated with a combination of chemotherapy and HER2-targeted monoclonal antibodies such as trastuzumab and pertuzumab. The HER2-targeted antibody-drug conjugates (ADCs) trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) represent a novel class of therapeutics in breast cancer. These drugs augment monoclonal antibodies with a cytotoxic payload, which is attached by a linker, forming the basic structure of an ADC. Novel combinations and sequential approaches are under investigation to overcome resistance to T-DM1 and T-DXd. Furthermore, the landscape of HER2-targeted therapy is rapidly advancing with the development of ADCs designed to attack cancer cells with greater precision and reduced toxicity. This review provides an updated summary of the current state of HER2-targeted ADCs as well as a detailed review of investigational agents on the horizon. Clinical trials are crucial in determining the optimal dosing regimens, understanding resistance mechanisms, and identifying patient populations that would derive the most benefit from these treatments. These novel ADCs are at the forefront of a new era in targeted cancer therapy, holding the potential to improve outcomes for patients with HER2-positive and HER2-Low breast cancer.

From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models

BackgroundNeuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent.MethodsWithin the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models.ResultsEighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as “very high priority”, that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient’s tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors.ConclusionsPREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

An Update on Current Therapeutic Options in IgA Nephropathy.

Immunoglobulin A nephropathy (IgAN) remains the leading cause of primary glomerular disease worldwide. Outcomes are poor with high rates of progressive chronic kidney disease and kidney failure, which contributes to global healthcare costs. Although this disease entity has been described, there were no disease-specific treatments until recently, with the current standard of care focusing on optimal supportive measures including lifestyle modifications and optimization of the renin-angiotensin-aldosterone blockade. However, with significant advances in the understanding of the pathogenesis of IgAN in the past decade, and the acceptance of surrogate outcomes for accelerated drug approval, there have been many new investigational agents tested to target this disease. As these agents become available, we envision a multi-pronged treatment strategy that simultaneously targets the consequences of ongoing nephron loss, stopping any glomerular inflammation, inhibiting pro-fibrotic signals in the glomerulus and tubulo-interstitium, and inhibiting the production of pathogenic IgA molecules. This review is an update on a previous review published in 2021, and we aim to summarize the developments and updates in therapeutic strategies in IgAN and highlight the promising discoveries that are likely to add to our armamentarium.

Toxicity-benefit analysis of advanced prostate cancer trials using weighted toxicity scoring.

110 Background: The weighted toxicity score (WTS) is a metric suitable for comparing the toxicity burden in experimental versus control arms of randomized controlled trials (RCTs). When compared against clinical endpoints, the WTS can be used to evaluate the cost versus benefit of anti-cancer agents. This study sought to apply the WTS to prostate cancer (PC) clinical trials. Methods: Select phase 3 PC RCTs with reported adverse event (AE) data were compiled. The WTS was computed for each trial arm and compared to reported hazard ratios (HRs) of primary and/or secondary endpoints (i.e., overall survival (OS) and progression-free survival (PFS)). Average WTSs for the experimental versus control arms were used to calculate the toxicity differential between treatment arms. Average HRs for OS and PFS were used to compare efficacy. Results: Sixteen RCTs were analyzed (investigational agents: androgen-receptor signaling inhibitors (ARSi) [n = 4], poly (ADP-ribose) polymerase inhibitors (PARPi) monotherapy [n = 3], ARSi + PARPi as well as ARSi + ARSi combination therapies [n = 3 each], and triple therapy [n=3]). The median number of distinct reported AEs across all studies was 18 (range, 9-61). Overall, toxicity and efficacy were greater among experimental than control arms (6.62 versus 4.00 median WTS; median HR for OS 0.71, median HR for PFS 0.61). The triple therapy studies observed the lowest increase in toxicity by adding an ARSi to control therapy (10.4%), associated with 52% lower risk of progression, and 29% lower risk of death. Comparably, the ARSi + PARPi trials noted the highest increase in toxicity (79.7%), with 32% lower risk of progression and 12% lower risk of death. The PARPi monotherapy, ARSi monotherapy, and ARSi + ARSi combination therapy studies reported increased toxicity due to experimental therapy in descending order (Table). Conclusions: The WTS enables toxicity versus benefit assessment for anti-cancer regimens. Clinical application of this metric may facilitate individualized treatment planning in advanced PC management. [Table: see text]

Impact of standard-of-truth method on evaluation of a diagnostic PET radiopharmaceutical: Learnings from the phase 3 SPOTLIGHT study.

39 Background: SPOTLIGHT (NCT04186845) evaluated newly approved diagnostic PET radiopharmaceutical 18F-flotufolastat (18F-rhPSMA-7.3) in patients (pts) with recurrent prostate cancer (PCa). In the absence of the gold standard histopathology, it can be challenging to verify positive findings from a diagnostic agent under investigation, particularly in recurrent PCa where it is neither feasible nor ethical to obtain multiple biopsies from the same patient. Consequently, conventional imaging is often used as a standard of truth (SoT) despite potential for it to be less sensitive than the investigational agent. Here, we explore the impact of SoT methods used in SPOTLIGHT on the study endpoints. Methods: The SPOTLIGHT coprimary endpoints were patient-level verified detection rate (VDR) and combined region-level positive predictive value (crPPV). Patient-level PPV was an exploratory endpoint. Pts with recurrent PCa underwent PET 50–70 min after IV administration of 296 MBq 18F-flotufolastat. For SoT assessment, the protocol required either histopathology or confirmatory imaging (≤60 and ≤90 days post-PET, respectively). Three blinded readers evaluated the scans, with majority read representing agreement between ≥2 readers. A separate Truth Panel established SoT for all pts. Post-study analysis revealed that, likely due to the study occurring during COVID, some pts had neither histopathology nor post-scan confirmatory imaging. These pts had only baseline/historic imaging as SoT. The present analysis stratified endpoint data according to the SoT method the panel used to verify PET-positive lesions. Results: Of 366 evaluable pts, 69 had histopathology and 297 had only confirmatory imaging for SoT. Of those with an imaging SoT, 90 (30%) had baseline/historic imaging only. As the Table shows, for each endpoint the highest value was obtained when using histopathology as SoT. VDR was ~4-fold higher with histopathology vs baseline/historic imaging, crPPV was 2-fold higher and PPV 2.5-fold. Conclusions: These data illustrate the substantial impact SoT methods can have on clinical trial endpoints and highlight a limitation of evaluating SPOTLIGHT endpoints in a cohort with various SoT methods (Table, column 1).1 Notably lower endpoint values are achieved when including pts with baseline/historic imaging SoT vs gold standard histopathology. 1. J Urol. 2023; 210:299-311. Clinical trial information: NCT04186845 . [Table: see text]

Advanced Practice Providers' Role in Intimacy in Advanced Cancer Patients.

The primary objectives of early phase clinical trials are to determine the safety, tolerability, and recommended doses of an investigational agent for patients with advanced cancer. Although these advances in cancer treatment have resulted in patients living longer, patients often experience psychological side effects that may affect sexual health and intimacy. It is estimated that cancer and its subsequent treatments affect the sexual health and intimacy of 40% to 100% of this patient population. Patients with advanced cancer who have undergone numerous treatments may be impacted negatively, not only physically (such as being disfigured by surgical resection) but also emotionally in relationship aspects with their intimate partners. Health-care providers who treat patients with advanced cancer enrolled in early phase clinical trials tend to focus primarily on treatment, physical side effects, and symptom management. Advanced practice providers (APPs) are accustomed to performing comprehensive physical examinations on patients; however, research indicates that intimacy and sexuality are most often excluded in discussions with patients with advanced cancer. The purpose of the article is to increase awareness of the need to address intimacy and sexuality in this patient population and to encourage APPs to make this a part of their everyday practice.

Quantitative MRI reveals heterogeneous impacts of treatment on diseased bone marrow in a mouse model of myelofibrosis.

Analyzing bone marrow in the hematologic cancer myelofibrosis requires endpoint histology in mouse models and bone marrow biopsies in patients. These methods hinder the ability to monitor therapy over time. Preclinical studies typically begin treatment before mice develop myelofibrosis, unlike patients who begin therapy only after onset of disease. Using clinically relevant, quantitative MRI metrics allowed us to evaluate treatment in mice with established myelofibrosis. We used chemical shift-encoded fat imaging, DWI, and magnetization transfer sequences to quantify bone marrow fat, cellularity, and macromolecular components in a mouse model of myelofibrosis. We monitored spleen volume, the established imaging marker for treatment, with anatomic MRI. After confirming bone marrow disease by MRI, we randomized mice to treatment with an approved drug (ruxolitinib or fedratinib) or an investigational agent, navitoclax, for 33 days. We measured the effects of therapy over time with bone marrow and spleen MRI. All treatments produced heterogeneous responses with improvements in bone marrow evident in subsets of individual mice in all treatment groups. Reductions in spleen volume commonly occurred without corresponding improvement in bone marrow. MRI revealed patterns associated with effective and ineffective responses to treatment in bone marrow and identified regional variations in efficacy within a bone. Quantitative MRI revealed modest, heterogeneous improvements in bone marrow disease when treating mice with established myelofibrosis. These results emphasize the value of bone marrow MRI to assess treatment in preclinical models and the potential to advance clinical trials for patients.

Abstract B003: Advancing biomarker discovery using a novel window of opportunity (WOO) trial for pancreatic ductal adenocarcinoma: Trial updates

Abstract Background: There are a growing number of targeted agents that have the potential to greatly improve the clinical outcomes of patients with PDAC beyond current standard of care chemotherapy. However, there is a lack in our understanding of which patients and intrinsic PDAC tumor types will best respond to a given targeted agent(s). In order to optimizing their clinical utility, it is crucial that we a deep understanding of the biological impact that these targeted agents (alone or combination) have within the human system. As such, we designed and implemented the WOO trial to provide a highly adaptable discovery platform for the preliminary assessment of biological activity of one or more targeted agents in patients with PDAC. Utilizing paired, pre- and on-treatment biopsies, along with a robust multiomics pipeline, the WOO trial enables independent assessment of the biological impact of targeted agent(s) on key signaling pathways that can be used to explore novel therapeutic strategies. Methods: WOO is a multi-arm early phase I trial platform designed to assess the pharmacodynamic (PD) effects of one or more study agent(s) alone or in combination in patients with PDAC. A Master Protocol is used to describe overarching design and logistics, and sub-protocols separately describe each study arm comprising the different investigational agent(s). Each participants undergoes a baseline tumor biopsy, then receives their assigned study agent(s) for a specified timeframe (not exceeding 30 days), before undergoing a repeated tumor biopsy, after which they continue to receive therapy per standard of care or clinical trials. Spatially-resolved, single cell multiplex assays are used to compare the effects of the study agent(s) on tumor cell state and heterogeneity between the paired samples. The primary study objective is to independently assess the PD feasibility of detecting a measurable change in tumor biology at post-treatment from baseline for participants within a study arm. The WOO trial uses a 2-stage Bayesian efficacy monitoring approach with a futility-stopping rule for each study arm. In stage 1 of each study arm, if 6 or more of the first 10 participants have a detectable change in tumor biology measurements (i.e., pre vs. post-treatment), then the study arm may continue to enroll an additional 10 participants. To date, 4 study arms are being evaluated: 1) poly (ADP-ribose) polymerase inhibitor (PARPi), olaparib (300 mg PO BID for 10 days), 2) MEK inhibitor (MEKi), cobimetinib (60 mg PO QD for 10 days), 3) ERK inhibitor, LY3214996 (400 mg PO QD for 10 days), and 4) PLK1 inhibitor, onvansertib (12 mg/m2 PO QD for 10 days). To date, a total of 34 participants have been enrolled and treated: n = 14 cobimetinib, n= 15 olaparib, n=2 onvansertib. Future arms to be added. Trial ID: NCT04005690. Citation Format: Charles D. Lopez, Adel Kardosh, Emerson Chen, Guillame Pegna, Alexander Guimaraes, Bryan Foster, Brian Brinkerhoff, Shaun M. Goodyear, Erin Taber, Brindha Rajagopalan, Johnson Vo, Kiara Siex, Brett Johnson, Danielle Galipeau, Dove Keith, Brett Sheppard, Brody Jonathan, Rosalie Sears, Gordon Mills. Advancing biomarker discovery using a novel window of opportunity (WOO) trial for pancreatic ductal adenocarcinoma: Trial updates [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B003.

A phase I dose-escalation study of pulsatile afatinib in patients with recurrent or progressive brain cancer.

Afatinib (BIBW2992; Gilotrif®) is a selective and irreversible inhibitor of the epidermal growth factor receptor (ErbB; EGFR) family. It inhibits EGFR, HER2, and HER4 phosphorylation, resulting in tumor growth inhibition and regression. This phase I dose-escalation trial of pulsatile afatinib examined the safety, drug penetration into the central nervous system, preliminary antitumor activity, and recommended phase II dose in patients with progressive or recurrent brain cancers. Afatinib was taken orally once every 4 days or once every 7 days depending on dose cohort, until disease progression or unacceptable toxicity. A total of 24 patients received the investigational agent and were evaluable for safety analyses, and 21 patients were evaluable for efficacy. Dosing was administered at 80mg every 4 days, 120mg every 4 days, 180mg every 4 days, or 280mg every 7 days. A recommended phase II dose of pulsatile afatinib was established at 280mg every 7 days as there were no dose-limiting toxicities in any of the dosing cohorts and all toxicities were deemed manageable. The most common drug-related toxicities were diarrhea, rash, nausea, vomiting, fatigue, stomatitis, pruritus, and limb edema. Out of the 21 patients evaluable for efficacy, 2 patients (9.5%) exhibited partial response based on Response Assessment in Neuro-Oncology criteria and disease stabilization was seen in 3 patients (14.3%). Afatinib taken orally was safe and well-tolerated up to 280mg every 7 days in brain cancer patients.

Becker Muscular Dystrophy: Could Altering the Natural History of Decline Help Tackle Unmet Medical Need?

This symposium was held as part of the 28th International Annual Congress of the World Muscle Society (WMS), held in Charleston, South Carolina, USA. Speakers reviewed the natural history of Becker muscular dystrophy (Becker), outlined the top line, 12-month data from the ARCH open label study of investigational agent EDG-5506, and aimed to put those results into clinical context. Becker is a serious rare disease with significant physical, emotional, financial, and social impact on the affected individuals and their caregivers. Once function begins to decline, males affected by the progressive X-linked genetic disorder continue to irreversibly lose muscle, ultimately leading to the loss of ambulatory and cardiopulmonary function. After discussing the aetiology of the condition, Erik Niks, Paediatric and Adult Neurologist, Leiden University Medical Center (LUMC), the Netherlands, presented the findings of natural history studies. They showed that while the age at which decline begins varies, once it does start, patients tend to experience a consistent decline in function equivalent to around 1.2–1.3 North Star Ambulatory Assessment (NSAA) points each year. This finding, combined with data on using MRI as a biomarker of disease progression, provides an evidence-based framework for clinical trial design, he argued. Sam Collins, Vice President of clinical development, Edgewise Therapeutics, Boulder, Colorado, USA, then presented topline 12-month data from the ARCH study. It found that EDG-5506 was well tolerated, and, importantly, recorded the stabilisation of functional assessments, including the NSAA, with a trend towards improvement, as well as rapid, sustained, and significant decreases in biomarkers of progression, including those related to muscle damage. Putting the ARCH study data into context, Barry Byrne, Director of the Health Center for Advanced Therapeutics and Powell Gene Therapy Center, University of Florida (UF), Gainesville, USA, explained exactly how declining NSAA status translated into life-altering function loss. Stabilising function, or even reducing the speed of decline, was an important goal for patients, he said, adding that meeting it could help to address significant unmet medical need.

Real world data on outcomes of anti-CD38 antibody treated, including triple class refractory, patients with multiple myeloma: a multi-institutional report from the Canadian Myeloma Research Group (CMRG) Database

Multiple myeloma (MM) remains incurable despite the availability of novel agents. This multi-center retrospective cohort study used the Canadian Myeloma Research Group Database to describe real-world outcomes of patients withanti-CD38 monoclonal antibody (mAb) refractory MM subsequently treated with standard of care (SoC) regimens. Patients with triple class refractory (TCR) disease (refractory to a proteasome inhibitor, immunomodulatory drug, and anti-CD38 mAb) were examined as a distinct cohort. Overall, 663 patients had disease progression on anti-CD38 mAb therapy, 466 received further treatment (346 with SoC regimens were included, 120 with investigational agents on clinical trial and were excluded). The median age at initiation of subsequent SoC therapy of 67.9 (range 39.6–89.6) years with a median of 3 prior lines (range 1–9). The median PFS and OS from the start of subsequent therapy was 4.6 (95% CI 4.1–5.6) months and 13.3 (95% CI 10.6–16.6) months, respectively. The median PFS and OS of patients with TCR disease (n = 199) was 4.4 (95% CI 3.6–5.3) months and 10.5 (95% CI 8.5–13.8) months. Our results reinforce that real-world patients with relapsed MM, particularly those with TCR disease, have dismal outcomes. There remains an urgent unmet need for the development of and access to effective therapeutics for these patients.

Model-based meta-analysis using latent variable modeling to set benchmarks for new treatments of systemic lupus erythematosus.

Several investigational agents are under evaluation in systemic lupus erythematosus (SLE) clinical trials but quantitative frameworks to enable comparison of their efficacy to reference benchmark treatments are lacking. To benchmark SLE treatment effects and identify clinically important covariates, we developed a model-based meta-analysis (MBMA) within a latent variable model framework for efficacy end points and SLE composite end point scores (BILAG-based Composite Lupus Assessment and Systemic Lupus Erythematosus Responder Index) using aggregate-level data on approved and investigational therapeutics. SLE trials were searched using PubMed and www.clinicaltrials.gov for treatment name, SLE and clinical trial as search criteria that resulted in four data structures: (1) study and investigational agent, (2) dose and regimen, (3) baseline descriptors, and (4) outcomes. The final dataset consisted of 25 studies and 81 treatment arms evaluating 16 different agents. A previously developed (K Goteti etal. 2022) SLE latent variable model of data from placebo arms (placebo + standard of care treatments) was used to describe aggregate SLE end points over time for the various SLE placebo and treatment arms in a Bayesian MBMA framework. Continuous dose-effect relationships using a maximum effect model were included for anifrolumab, belimumab, CC-220 (iberdomide), epratuzumab, lulizumab pegol, and sifalimumab, whereas the remaining treatments were modeled as discrete dose effects. The final MBMA model was then used to benchmark these compounds with respect to the maximal efficacy on the latent variable compared to the placebo. This MBMA illustrates the application of latent variable models in understanding the trajectories of composite end points in chronic diseases and should enable model-informed development of new investigational agents in SLE.