Single-agent Efficacy Research Articles

Abstract 3914: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target

Abstract Background: Loss of MHC-I antigen presentation is often associated with T-cell excluded tumors, and represents a common mechanism of both primary and acquired resistance to immune checkpoint blockade (ICB) treatment. MHC-I normally acts as a marker of “self” and cells that lack MHC-I are recognized and killed by innate immunity. In this study, we investigated the mechanism of innate immune evasion in tumors with MHC-I antigen presentation defects. Methods: Integrating functional genomics, big data, and artificial intelligence, we discovered that IGSF8 expressed on cancer cells is an evolutionarily conserved innate immune checkpoint. IGSF8 is normally expressed in neuronal tissues and is not essential in vitro or in vivo. However, knockout of IGSF8 in B16-F10 melanoma cell line decreased tumor growth in vivo. For clinical relevance of IGSF8 in tumor immunity, we analyzed genomics, transcriptomics, and clinical data from The Cancer Genome Atlas (TCGA). We developed an antibody (GV20-0251) against IGSF8 and tested its function to induce immune cell killing of cancer cells in vitro and inhibit tumor growth in vivo. Results: IGSF8 has receptors on both natural killer (NK) cells and dendritic cells (DC) to strongly suppress NK cytotoxicity and antigen presentation. In the TCGA tumor profiles, IGSF8 has highest mRNA expression in melanoma and is significantly overexpressed in many cancer types. IGSF8 also shows frequent copy number amplifications. In addition, IGSF8 expression is associated with low antigen presentation, low immune infiltration, and worse overall survival in patients with low MHC class I expression. Immunohistochemistry staining of various cancers showed that IGSF8 expression was primarily observed in malignant cells with low MHC-I. We developed a cross-species reactive antibody against IGSF8 (GV20-0251) which blocks IGSF8 interaction with NK and DC. Flow cytometry of the tumor infiltrating CD45+ cells revealed that IGSF8 inhibition significantly increased NK and dendritic cell infiltration. GV20-0251 enhances NK killing of cancer cells in vitro and increases antigen presentation, NK-mediated cytotoxicity, and T cell signaling in vivo. In multiple syngeneic tumor models (B16-F10, CT26, LLC, 4T1 and EMT6), anti-IGSF8 shows single-agent efficacy and is synergistic with anti-PD1 in controlling tumor growth. Conclusions: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target. A phase 1 study is ongoing to explore the IGSF8 inhibitor GV20-0251 in patients with advanced or metastatic solid tumors (NCT05669430). Citation Format: X. Shirley Liu, Yulong Li, Xiangyang Wu, Hailing Liu, Huizhu Liu, Caibin Sheng, Qingyang Ding, Yixuan Tang, Chao Liu, Bin Xie, Xi Xiao, Quan Yu, Rongbin Zheng, Xihao Hu, Tengfei Xiao. IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target [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 3914.

Abstract 6510: BPI-511966,a potent and selective HPK1 inhibitor with robust single-agent efficacy for cancer immunotherapy

Abstract Although immune checkpoint inhibitors (ICIs) have become the standard treatment for many types of cancers, a large proportion of patients fail to respond to ICIs. Hematopoietic progenitor kinase 1 (HPK1) has been validated as a negative intracellular immune checkpoint that constrains T-cell activation. Genetic or pharmacological ablation of HPK1 induces a robust anti-cancer immune response in vivo, highlighting the potential of HPK1 as a next-generation target for cancer immunotherapy. Herein, we reported the discovery of BPI-511966, a highly potent ATP-competitive HPK1 inhibitor. The compound strongly inhibited HPK1 kinase with an IC50 of 2.3 nM at physiologically relevant ATP concentration (1 mM) and reduced phosphorylation of SLP-76, a substrate adaptor of HPK1, in Jurkat T cells in vitro, with an IC50 value of 11 nM. Development of an optimal HPK1 inhibitor requires not only high potency for target engagement but also high selectivity to spare other kinases that are essential for T cell activation. BPI-511966 showed >100-fold higher IC50 (364 nM) against MAP4K3/GLK, an HPK1 homology kinase promoting T-cell activation, and also demonstrated weak inhibition in a panel of other immune-related kinases. The remarkable potency and selectivity of BPI-511966 translate into robust T cell-enhancing efficacy in vitro. Treatment with BPI-511966 promoted both IL-2 and IFN-γ production by Jurkat T cell and human PBMC in a dose-dependent manner. The on-target action of BPI-511966 was validated in HPK1-KO Jurkat T cells, in which no significant impacts on SLP-76 phosphorylation and IL-2 secretion were observed. In vivo, single dose escalation of BPI-511966 in mice leads to dose-proportional increases in drug exposure, accompanied by inhibition of SLP-76 phosphorylation in peripheral CD3+ T cells. Furthermore, repeated oral administration of BPI-511966 as a single agent significantly suppressed CT-26 tumor growth in immune-competent mice (92% TGI), correlating with increased infiltration of CD45+ CD8+ cells and enhanced cytokine production in tumor. Notably, the efficacy of BPI-511966 is abrogated in immune-compromised mice, suggesting its vital role in promoting anti-cancer immunity. In summary, this study presents a novel, highly potent, and selective HPK1 inhibitor BPI-511966. The robust single-agent activity of BPI-511966 makes it a promising small molecule I/O agent to address ICI resistance as monotherapy or in combination. Citation Format: Yong Zhang, Xiaoguan Zhu, Wenmao Wu, Zhifang Xia, Zhengyao Zou, Han Han, Cheng Yang, Haibo Chen, Tianyi Ma, Xiaoyun Liu, Jing Guo, Hao Wu, Hong Lan, Lieming Ding, Jiabing Wang, Quan Zhou. BPI-511966,a potent and selective HPK1 inhibitor with robust single-agent efficacy for cancer immunotherapy [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 6510.

Abstract 514: Salt-inducible kinase inhibitor OMX-0407 drives cell-cycle arrest in vitro and in vivo: An in-depth MoA analysis by phospho-proteomics

Abstract Based on iOmx’ proprietary iOTargTM genetic screening platform, salt-inducible kinase (SIK) 3 was identified as a novel cell signaling modulator in cancer biology. OMX-0407, an orally available, spectrum-selective kinase inhibitor targets several members of the SIK family and is known to prevent tumor-promoting function of the SIK-family through repolarization of the tumor microenvironment by enhancing caspase-mediated apoptosis upon death-receptor signaling. In addition, OMX-0407 inhibits individual key members of the tyrosine and tyrosine-like kinase family that are involved in cancer cell proliferation and cell cycle regulation. Via this dual mode of action, OMX-0407 has the potential to effectively fight solid tumors as exemplified by its striking single-agent efficacy in multiple pre-clinical tumor models. A comprehensive anti-tumor viability screen of >200 human cancer cell lines revealed striking effects of OMX-0407 on cancer cell viability across various cancer indications, resulting in a distinct sensitivity profile across various tumor indications with particularly strong effects on renal cell carcinoma (RCC) and squamous non-small cell lung cancer (sqNSCLC). Phospho-proteomics screening demonstrated pharmacodynamic activity of OMX-0407 in sensitive tumor cell lines via inhibition of key cellular processes such as cell motility, proliferation, and cell cycle regulation across different indications. Orthogonal functional in vitro assays confirmed the association of the OMX-0407-mediated anti-tumor efficacy with the arrest of G1/S transition and corresponding downregulation of cell cycle associated proteins PAK1/2 and ARHGAP35. The profound impact of OMX-0407 on fundamental regulatory mechanisms of cell division and cancer cell apoptosis translates into dose-dependent single-agent, anti-tumor efficacy in various pre-clinical tumor models of selected indications. Combination with Axitinib, a selective orally available inhibitor of vascular endothelial growth factor receptor 2, significantly enhanced anti-tumor efficacy and pharmacodynamic inhibition of cell growth as well as angiogenesis. These data further strengthen the potential of OMX-0407 in the treatment of RCC as well as other indications. In summary, OMX-0407 is a novel spectrum selective kinase inhibitor, currently under evaluation in a clinical Phase I trial (NCT05826600). OMX-0407 demonstrates strong anti-tumor efficacy in monotherapy in selected sensitive indications through a dual-pronged MoA, by modulating the tumor microenvironment and exerting direct anti-tumor activity in solid tumor indications with high unmet medical need. Citation Format: Ilona-Petra Maser, Marisa Stebegg-Wagner, Bettina Bauer, Filippos Konstantinidis, Andreas Schirmer, Moritz Zulley, Sonja Lacher, Barbara Kracher, Parastou Kohvaei, Murray Yule, Hannes Loferer, Stefan Bissinger. Salt-inducible kinase inhibitor OMX-0407 drives cell-cycle arrest in vitro and in vivo: An in-depth MoA analysis by phospho-proteomics [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 514.

CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy

AbstractEffective T-cell responses not only require the engagement of T-cell receptors (TCRs; “signal 1”), but also the availability of costimulatory signals (“signal 2”). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell–mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19–4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

Comparison of efficacy and tolerability of single agent and double agent chemotherapy regimens in first-line treatment of elderly patients with her-2 negative metastatic gastric cancer

Aims: Chemotherapy remains a cornerstone in treating metastatic gastric cancer (GC), yet the management of elderly patients, who often face distinct challenges, lacks comprehensive guidelines. The aim of this study was to compare the efficacy and side effects of single-agent and double-agent chemotherapy regimens in first-line treatment of elderly patients with HER-2 negative metastatic GC. Methods: We retrospectively evaluated HER-2 negative metastatic GC patients aged 80 years and older who were treated at Van Yüzüncü Yıl University Medical Faculty Dursun Odabaşı Medical Center Oncology Clinic between 2010 and 2023. Demographic characteristics, treatment regimens and responses, prognostic factors, grade 3-4 toxicity, progression-free survival (PFS), and overall survival (OS) were analyzed. Results: The mean age of 56 patients was 82.6 ± 2.3 years and 24 (42.9%) of them were women. Single-agent chemotherapy was administered to 33 (58.9%) patients, while 23 (41.1%) received double-agent chemotherapy. The median OS was 5 months (95% CI, 2.9 to 7.1) in the single-agent group and 10 months (95% CI, 4.2 to 15.8) in the double-agent group (p = 0.237), although there was a numerical difference, it was not statistically significant. Median PFS was longer with double-agent chemotherapy, but not statistically significant (6 months vs. 4 months, p = 0.668). No statistically significant difference was found in the side effect rates of patients receiving single and double-agent chemotherapy. Conclusion: In our study, despite the absence of statistical significance in the survival rates among patients receiving double chemotherapeutic agents, their survival was twice as long as that of individuals receiving a single agent. Furthermore, no significant differences in terms of side effects were observed. These findings suggest that, even in individuals aged 80 years and older, a preference for double-agent chemotherapy should be considered when feasible.

Therapeutic Discovery for Chromatin Complexes: Where Do We Stand?

In this review, we explore the current landscape of preclinical and clinical therapeutics targeting epigenetic complexes in cancer, focusing on targets with enzymatic inhibitors, degraders, or ligands capable of disrupting protein–protein interactions. Current strategies face challenges such as limited single-agent clinical efficacy due to insufficient disruption of chromatin complexes and incomplete dissociation from chromatin. Further complications arise from the adaptability of cancer cell chromatin and, in some cases, dose-limiting toxicity. The advent of targeted protein degradation (TPD) through degrader compounds such as proteolysis-targeting chimeras provides a promising approach. These innovative molecules exploit the endogenous ubiquitin–proteasome system to catalytically degrade target proteins and disrupt complexes, potentially amplifying the efficacy of existing epigenetic binders. We highlight the status of TPD-harnessing moieties in clinical and preclinical development, as these compounds may prove crucial for unlocking the potential of epigenetic complex modulation in cancer therapeutics.

Response-guided neoadjuvant sacituzumab govitecan for localized triple-negative breast cancer: results from the NeoSTAR trial

Sacituzumab govitecan (SG), a novel antibody-drug conjugate (ADC) targeting TROP2, is approved for pre-treated metastatic triple-negative breast cancer (mTNBC). We conducted an investigator-initiated clinical trial evaluating neoadjuvant (NA) SG (NCT04230109), and report primary results. Participants with early-stage TNBC received NA SG for four cycles. The primary objective was to assess pathological complete response (pCR) rate in breast and lymph nodes (ypT0/isN0) to SG. Secondary objectives included overall response rate (ORR), safety, event-free survival (EFS), and predictive biomarkers. A response-guided approach was utilized, and subsequent systemic therapy decisions were at the discretion of the treating physician. From July 2020 to August 2021, 50 participants were enrolled (median age= 48.5 years; 13 clinical stage I disease, 26 stage II, 11 stage III). Forty-nine (98%) completed four cycles of SG. Overall, the pCR rate with SG alone was 30% [n= 15, 95% confidence interval (CI) 18% to 45%]. The ORR per RECIST V1.1 after SG alone was 64% (n= 32/50, 95% CI 77% to 98%). Higher Ki-67 and tumor-infiltrating lymphocytes (TILs) were predictive of pCR to SG (P= 0.007 for Ki-67 and 0.002 for TILs), while baseline TROP2 expression was not (P= 0.440). Common adverse events were nausea (82%), fatigue (76%), alopecia (76%), neutropenia (44%), and rash (48%). With a median follow-up time of 18.9 months (95% CI 16.3-21.9 months), the 2-year EFS for all participants was 95%. Among participants with a pCR with SG (n= 15), the 2-year EFS was 100%. In the first NA trial with an ADC in localized TNBC, SG demonstrated single-agent efficacy and feasibility of response-guided escalation/de-escalation. Further research on optimal duration of SG as well as NA combination strategies, including immunotherapy, are needed.

Abstract A099: Best in class, potent, SOS1 inhibitors demonstrate single agent activity in preclinical models of KRAS driven tumors

Abstract KRAS is an oncogene implicated in a wide variety of tumors (~21% of solid tumors harbor KRAS mutations). Interaction of KRAS with its predominant Guanine Exchange Factor (GEF), SOS1, is crucial for activation of KRAS and further KRAS mediated oncogenic signaling. Thus, pharmacological inhibition of SOS1 is expected to be effective in treating RAS driven cancers. Biochemical potency of compounds was confirmed in a SOS1 mediated FRET based guanine exchange factor (GEF) assay. Multiple potent SOS1 inhibitors were identified with potencies in the low double digit nanomolar range. Selectivity was determined against SOS2 and a host of other kinases in the RAS/RAF/MEK or RAS/PI3K/AKT pathways. Anti-proliferative activity across a panel of WT and mutant KRAS cell lines revealed IC50 between 50-200 nM with synergism observed when combined with MAPK pathway inhibitors as well as the KRAS G12C inhibitor, Sotorasib, in a 3D anchorage independent assay format. Correspondingly, significant reduction in PD biomarkers (>50% at 100 nM), pERK and pAKT, was demonstrated in KRAS mutant as well as EGFR/PI3K mutant cell lines. PK-PD correlation was also established in a tumor bearing mice model, with dose-dependent reduction of both pERK and pAKT. Compounds demonstrated excellent ADME properties with significant tissue distribution upon oral dosing. One of the lead molecules demonstrated significant single agent efficacy with growth inhibition of approximately 70% in a KRAS G12C bearing NSCLC xenograft model. No adverse signs including reduction in food intake or body weight loss were reported during the study period. IND-enabling studies are currently ongoing for the lead compounds. Citation Format: Balakrishna Lakhavath, Manoj Pothuganti, Githavani Kummari, Hemasankar Pathange, Rajan Sekar, Srikant Viswanadha. Best in class, potent, SOS1 inhibitors demonstrate single agent activity in preclinical models of KRAS driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A099.

Abstract C150: Targeted inhibition of homologous recombination and nonhomologous end joining in diffuse intrinsic pontine gliomas to prevent tumor recurrence

Abstract Therapeutic resistance remains a major obstacle to successful clinical management of Diffuse Intrinsic Pontine Glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. Based on these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrate that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Since radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of ATM to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease. Citation Format: Ivana Barravecchia, Monika Sharma, Robert Teis, Jeanette Cruz, Rachel Mumby, Elizabeth Ziemke, Carlos Espinoza, Brian Magnuson, Mats Ljungman, Carl Koschmann, Christopher Whitehead, Judith Sebolt-Leopold, Stefanie Galban. Targeted inhibition of homologous recombination and nonhomologous end joining in diffuse intrinsic pontine gliomas to prevent tumor recurrence [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C150.

Abstract B012: Validation of the OncoSignature assay, an ACR-368-tailored response-predictive quantitative multiplexed immunofluorescent assay for prediction of sensitivity to the CHK1/2 inhibitor ACR-368 in individual patients with cancer

Abstract Background: The CHK1/2 kinase inhibitor ACR-368 (Prexasertib) demonstrated durable single-agent efficacy in a subset of patients with high grade serous ovarian, anal and head and neck cancers in past trials. Using a quantitative phospho-proteomics approach, we identified three orthogonal candidate biomarkers tailored to predict tumor response to ACR-368 treatment. The OncoSignature® assay is based on immunofluorescent staining of biomarkers in FFPE tumor sections with image acquisition, analysis, and scoring of biomarkers. The sample is OncoSignature positive if all three biomarker scores are above their thresholds. We validated the OncoSignature® assay to predict response to ACR-368 in cancer cell lines and ovarian cancer PDX models. Here, we further validated OncoSignature® assay in novel cancer indications and in patient biopsy samples from ACR-368 clinical trials. Methods: Multiple cancer types were tested with the OncoSignature® assay for prevalence of potential ACR-368 responders. For validation, endometrial cancer PDX models were tested in anACR-368 efficacy study. Blinded untreated PDX tumors were analyzed in the OncoSignature® assay and results locked prior to unblinding and statistical analysis. Adapted [CS1] Oncosignature assay was applied to frozen pretreatment ovarian cancer biopsies from a single center ACR-368 monotherapy Ph2 trial (NCT02203513). OncoSignature® assay was also applied to FFPE pretreatment ovarian cancer biopsies from a multi-center ACR-368 monotherapy Ph 2 trial (NCT03414047) and from the NCT02203513 trial. After biomarker scores were generated and locked, patient treatment outcomes were unblinded and ACR-368 response was compared with OncoSignature prediction. Results: Evaluation of multiple cancer indications revealed a concordance between OncoSignature-predicted prevalence of ACR-368 responders and results of ACR-368 treatment observed in clinical trials. Metastatic ovarian cancer had 30% of predicted responders and 30% and 14% observed RECIST responses the above-cited trials. Squamous lung cancer had no predicted responders and no RECIST responses in a clinical trial. Endometrial and bladder cancer had a high prevalence of predicted responders with 40% and 36%, respectively. The endometrial cancer PDX efficacy study had multiple complete responses predicted by OncoSignature® assay with an AUC of 0.88. For frozen and FFPE patient biopsy samples from Phase 2 ACR-368 trials, OncoSignature positive groups had highly significant response enrichment to 47% and 57%, respectively, and significant increases in progression free survival time. Conclusions: Bladder and endometrial cancers were identified as novel indications with high prevalence of predicted ACR-368 responders. The OncoSignature® assay significantly predicted response in endometrial PDX models and in samples from ovarian cancer patients treated with ACR-368. OncoSignature® assay driven clinical trial (NCT05548296) in ovarian, endometrial and bladder cancers was initiated to test benefit to patients. Citation Format: Ayesha Murshid, Christina Noe, Kailash Singh, Sibgat Choudhury, Jayakumar Nair, James Dunyak, Michail Shipitsin, Jung-Min Lee, Peter Blume-Jensen. Validation of the OncoSignature assay, an ACR-368-tailored response-predictive quantitative multiplexed immunofluorescent assay for prediction of sensitivity to the CHK1/2 inhibitor ACR-368 in individual patients with cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B012.

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status.

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor-initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 (Son of Sevenless 1) or KSR1 (Kinase Suppressor of RAS 1) both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid-initiating cells in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA comutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wild-type cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS-mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying comutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

Preclinical Characterization of AZD9574, a Blood-Brain Barrier Penetrant Inhibitor of PARP1.

We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.

EXTH-32. SYNERGISTIC EFFICACY OF Y BOX BINDING PROTEIN 1 (YBX1) INHIBITION COMBINED WITH CHEMOTHERAPY FOR THE TREATMENT OF MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract Medulloblastoma is the most common pediatric brain tumor in children. Patients diagnosed with MYC-amplified group 3 (G3) have a dismal prognosis with a 5 year survival rate of < 50%. Standard of care consists of maximal resection surgery followed by craniospinal irradiation and chemotherapy, but patients experience diminished quality of life from these extensive treatment regimens. Recent observations have shown that Y-box binding protein 1 (YBX1) is upregulated across human medulloblastoma subgroups compared to normal cerebellum. G3 patient tumors have the highest expression of YBX1, possibly due to an oncogenic feedback loop. We utilized an orthogonal pharmacogenetic approach to target YBX1. Knockdown/out of YBX1 significantly inhibits proliferation of G3 MB cells in vitro and in vivo. We next investigated the therapeutic potential of a novel YBX1 inhibitor, which displays single agent efficacy at decreasing the proliferation of medulloblastoma cell line derived xenografts (CDX). Since YBX1 targeting downregulated ABC transporter expression, we hypothesize that inhibition of YBX1 may sensitize medulloblastoma cells to chemotherapeutic agents. We performed drug synergy experiments combining YBX1 inhibition and commonly used standard of care chemotherapy. We identify that combining YBX1 inhibition sensitizes to both gemcitabine and vincristine in vitro and in vivo. We are currently evaluating this YBX1 inhibitor in an “anchor-probe” screen combining with all currently FDA approved drugs. Our findings impart an alluring prospect of targeting YBX1 in combination with chemotherapy, and possibly other compounds could improve patients’ outcomes.

DDDR-05. TARGETED INHIBITION OF DNA REPAIR AND SURVIVAL SIGNALING IN DIFFUSE INTRINSIC PONTINE GLIOMAS

Abstract Therapeutic resistance remains a major obstacle to successful clinical management of Diffuse Intrinsic Pontine Glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. Based on these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrate that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Since radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of ATM to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

Loncastuximab Tesirine Demonstrated Substantial Single-Agent Efficacy and Manageable Safety Profile in Heavily Pretreated Chinese Patients with Relapsed or Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Patients with R/R DLBCL who failed multi-agent chemoimmunotherapies usually have a poor prognosis, leaving a significant unmet medical need. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate, composed of a humanized anti-CD19 antibody conjugated to a pyrrolobenzodiazepine dimer toxin that showed clinical efficacy and manageable safety in heavily pretreated DLBCL patients in a global phase 2 study of Lonca monotherapy (NCT03075696). Here we present results from a phase 2 study (ChiCTR2300072058) of Lonca monotherapy in Chinese patients with R/R DLBCL for the first time, including analyses of responses in high-risk DLBCL subgroups. Methods: This open-label, single-arm, phase 2 study was designed to confirm the safety, efficacy and PK profiles of Lonca monotherapy in Chinese patients with R/R DLBCL who had failed ≥ 2 lines of systemic therapies. The primary endpoint was overall response rate (ORR) as assessed by IRC according to the Lugano 2014 criteria. Secondary efficacy endpoints include complete response rate (CRR), DOR, RFS, PFS, and OS. Treatment-emergent adverse events (TEAEs) were reported by CTCAE v4.0. Results: As of data cutoff (11-Jan-2023), 64 Chinese patients with DLBCL were enrolled and received at least one dose of Lonca (median: 4.0 cycles [range: 1-17]) and were evaluable for safety and efficacy analyses. The median number of prior lines of therapies was 3.0 (range: 2-12), with 67.2% of patients having ≥ 3 lines of prior therapies. Four patients (6.3%) had received prior CD19 CAR-T therapy. Forty-three patients (67.2%) were refractory to the first-line therapy (primary refractory), 56 patients (87.5%) were refractory to the most recent line of therapy and 40 patients (62.5%) were refractory to all prior lines of therapies. At data cutoff, the median follow-up was 8.5 months. The ORR by IRC was 51.6% (95% CI: 38.7% to 64.2%), and CRR was 23.4%. The median DOR (mDOR) was 6.37 months as assessed either by IRC or by investigator. For patients with a CR, the mDOR was 10.22 months (6.08 months for patients with PR). The median time to first response was 41.0 days. The median PFS and median OS were 4.96 and 9.33 months, respectively. Responses, including CRs, were observed in several high-risk subgroups (Fig. 1): ORR was 54.7% in patients with advanced stage disease (Stage III/IV) and 50.0% in patients with transformed disease. Patients who were refractory to first-line therapy, to the most recent therapy, or to all prior therapies had ORRs of 44.2%, 51.8% and 42.5%, respectively. Three patients who had received a prior SCT had an ORR of 100%. Lonca was also effective in elderly patients ≥ 65 years (ORR of 45.8%, mDOR of 6.37 months) and in patients who had received prior CD19 CAR-T therapy (ORR of 50%). Overall, 64 (100%) patients had at least one TEAE and 61 (95.3%) patients had at least one TEAE ≥ Grade 3. The most common (≥30%) all-grade TEAEs were GGT increased (71.9%), followed by anaemia (70.3%), platelet count decreased (65.6%), AST increased (64.1%), WBC count decreased (64.1%), neutrophil count decreased (60.9%), ALT increased (51.6%), hypokalaemia (37.5%), and blood ALP increased (32.8%). The most common (≥15%) TEAEs of Grade ≥3 were platelet count decreased (34.4%), neutrophil count decreased (28.1%), WBC count decreased (28.1%), GGT increased (25.0%), anaemia (18.8%), hypokalaemia (18.8%), neutropenia (17.2%), and lymphocyte count decreased (15.6%). The rate of febrile neutropenia was low (3.1%). The majority of Grade ≥ 3 TEAEs were abnormalities of clinical laboratory values as opposed to clinical symptoms and were generally reversible. Treatment-related TEAEs leading to treatment discontinuation occurred in 10 patients (15.6%), most commonly GGT increased (3 patients; 4.7%). No increase in toxicity was seen in patients aged ≥65 years compared with younger patients. Conclusions: Lonca demonstrated substantial and clinically meaningful single-agent efficacy and was well-tolerated in heavily pretreated Chinese patients with R/R DLBCL. Toxicities were generally manageable and reversible in most patients by routine clinical practice and/or with dose modifications, with neither unexpected safety concerns nor different toxicity profile in patients aged ≥65 years. Encouraging and durable responses were also observed in high-risk patient groups, including patients with advanced stage, transformed or refractory DLBCL.

Dual Targeting of ROR1 and BTK Augments the Anti-Lymphoma Activity in Mantle Cell Lymphoma

Background Mantle cell lymphoma (MCL) is a rare and aggressive subtype of non-Hodgkin lymphoma. Development of BTK inhibitors (BTKis) and CAR-T cells revolutionized the treatment paradigm for the patients with MCL. However, this aggressive B-cell lymphoma frequently relapses leading to poor survival. Therefore, novel therapies are in urgent need to overcome the therapeutic resistance. ROR1 is normally expressed during embryonic development but is usually absent or expressed at low levels in adult tissues. However, re-expression of ROR1 was found in various types of cancer, especially in cancer stem/tumor-initiating cells. ROR1 persistently activates oncogenic signaling pathways in the BCR/BTK-independent manner. Accordingly, we hypothesize that dual targeting of ROR1 and BTK may augment the therapeutic efficacy, and thus combination of monoclonal antibody zilovertamab (cirmtuzumab) targeting ROR1, and BTKis will overcome the therapeutic resistance and effectively kill the relapsed/refractory MCL cells. Methods A panel of MCL cell lines and primary patient samples were used in this study. ROR1 expression levels in MCL cell lines and primary MCL samples were determined by flow cytometry and western blotting. For establishment of patient-derived organoid (PDO), the patient samples or MCL cell from PDX tumors were resuspended in culture medium containing cytokine cocktails. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. The established PDOs were applied for drug sensitivity assays. In addition, a patient-derived xenograft (PDX) mouse model was also used for evaluating single agent and combination efficacy of BTKis and zilovertamab (Zilo). Cell viability assays were performed using CellTiter routine or 3D cell viability assay kit (Promega). Results We hypothesize dual targeting of both BTK and ROR1 signaling pathways will augment cytotoxic effect on the MCL cells including major proliferative and minor ROR1 +CD117 + population, whose survival is not dependent on BTK, in tumor mass. To test the hypothesis, we first analyzed the ROR1 expression in MCL cell lines, primary and PDX MCL cells using flow cytometry and immunoblotting analysis. Our results indicated that in comparison with normal PBMC, ROR1 expression were detected in all examined MCL cell lines including JeKo-1, Mino, Z-138, Granta519, JVM2, SP49, Maver-1 and Rec-1. To test whether p53 status relates to sensitivity of MCL cells to BTKi and Zilo, we examined cytotoxic effect of combination of BTKi and Zilo on MCL cell lines (1) harboring mutated TP53 (T P53 Mut): JeKo-1, JeKo-R, Mino, and Mino-R and (2) harboring wild-type TP53 ( TP53 WT): Z-138 and Maver-1, and PDO as well as PDX tumors derived from biopsies bearing either TP53 WT or TP53 Mut. Treatment with single BTKi at 2-10 µM (Ibrutinib, zanubrutinib, acalabrutinib or pirtobrutinib) significantly induced cytotoxicity in the TP53 MutMCL cells, and the cell death was remarkedly increased when BTKi was combined with Zilo at 25-50 µg/ml. In contrast, in MCL cell lines with TP53 WT, BTKi-Zilo combination did not significantly result in increase of cytotoxic effect compared to single agents. These results suggest that the genetic status of TP53 is important for the augmented cytotoxic effect of BTKi-Zilo combination on MCL cells. To further evaluate potentiated effect of BTKi-Zilo combination, we performed ex vivo viability assay using PDO platform. Typically, PDOs were treated with either single agent or combined regimens. Consistent with the data in MCL cell lines, BTKi-Zilo combination resulted in potentiated cytotoxic activity in the PDO models derived from naïve and relapsed patients harboring TP53 Mut. Furthermore, BTKi-Zilo combination displayed synergistic effect in a PDX model derived from a patient harboring TP53 Mut. Conclusion Dual targeting of BTK and ROR1 signaling pathways augmented efficacy selectively in preclinical MCL models with TP53 Mut. These data provide insights to develop tailored therapeutics to improve patient outcome for patients with TP53 mutation.

Infection Risk and Use of Prophylaxis with Anti-BCMA Bispecific T Cell Engagers and Belantamab Mafodotin for Patients with Relapsed and Refractory Multiple Myeloma

Introduction: BCMA targeted therapies have demonstrated single agent efficacy for patients with triple class exposed or refractory multiple myeloma (RRMM). Teclistamab and Belantamab Mafodotin (Belamaf) are currently approved for RRMM in Europe with several Bispecific T Cell Engagers (TCE) under investigation. However, whilst TCEs have higher response rates and progression free survival to Belamaf, adverse events, particularly infections may influence treatment choice particularly for frailer patients. Intravenous Immunoglobulin (IVIg) is recommended to prevent infections once IgG levels fall <4g/L or even as prophylaxis with anti-BCMA TCEs. Methods: This was a retrospective review of MM patients treated with anti-BCMA monotherapies at University College London Hospitals between 28/07/2015 and 15/06/2023. IVIg was given according to UK guidelines: IgG of <3g/L and recurrent infections/ one severe infection, despite 6 months of prophylactic antibiotics. IgG levels between 3-4g/L also required failure of a vaccine challenge. IVIg for asymptomatic patients was not permitted. Statistics was performed using GraphPad Prism. Results: 55 patients were treated with anti-BCMA monotherapies: 21 with TCE and 34 with Belamaf. The median age was 61 years (range 45-79), 43% female, 80% white and 5.6% Black. The median time from diagnosis to treatment was 5.8 years with a median of 5 prior lines (range 2-15). 51.9% of patients had high risk cytogenetics. Patients were a median of 59 years for TCE (range 47-73) vs 64 years (range 45-79) with Belamaf. Both groups had a median of 5 prior lines and a median Charlson co-morbidity index of 2, but 40% of TCE were classified frail by modified IMWG score vs 61.8% with Belamaf. CD38 exposure was 100% for TCE patients and 76% Belamaf. Severe hypogammaglobulinemia (<4g/L) at baseline prior to anti-BCMA treatment was present in 75% of TCE and 53% of Belamaf patients. Further significant reductions in median IgG levels occurred during treatment (TCE: 3.04g/L to 1.06g/L, p=0.01; Belamaf: 3.9g/L to 1.9g/L p=0.002). Nadir IgG levels were significantly lower with TCE than Belamaf (p=0.047). All patients received aciclovir prophylaxis, PJP prophylaxis (co-trimoxazole/ azithromycin) was given in 100% with TCE and 82% with Belamaf, with anti-fungal prophylaxis in 1 TCE patient. IVIg was used prior to anti-BCMA treatment in 3 patients (2 TCE, 1 Belamaf). Whilst on anti-BCMA treatment, IVIg was used in 38% for TCE and 5.9% for Belamaf. The risk for all grade infection per patient was 3.7 times higher for TCE vs Belamaf (increased risk TCE vs Belamaf: G1-2: 3.3, G3: 2.1, G4: 2.1). There was a total of 104 infection episodes, 57% with TCE, 43% with Belamaf (see Figure). The median time to first infection was 45 days for TCE and 34 days for Belamaf. Most infections were low grade with 20 patients having ≥1 infection (10/21 (48%) TCE, 10/34 (29%) Belamaf). At Grade 1-2, there were 1.8 infections per patient for TCE vs 0.7 for Belamaf. For G3-5, 1.0 infections per patient vs 0.6 for Belamaf. There was 1 grade 5 infection with TCE, 0 for Belamaf. Respiratory infections predominated (TCE: 80% vs Belamaf: 73%) followed by urinary (TCE: 5% vs Belamaf 16%), blood (TCE 10% vs Belamaf 5.4%) and GI (TCE 3% vs Belamaf 5%). 96/ 104 infections were identified, most were viral (TCE 71% vs Belamaf 62%) with few fungal infections (6% vs 4%). More bacterial infections occurred with Belamaf possibly due to lower antibacterial prophylaxis and IVIg use (TCE: 23% vs Belamaf: 35%). Since commencing IVIg, 5 of 8 (63%) TCE patients had at least 1 infection, mainly viral. A median of 1 further infection (range 0-14) occurred over a median of 3.2m (range 0.6-28.3m) of monthly IVIg infusions. Routine blood viral PCR monitoring for CMV, EBV & Adenovirus was performed in 30% of patients. 13 (65%) of TCE patients had 197 blood viral PCR tests performed of which 3 patients (23%) tested positive (2x adenovirus and 1 CMV). Of these 2 (both adenovirus) had symptoms requiring intervention. Of the 8.8% of Belamaf patients tested, 0 were positive. Conclusions: Patients treated with Belamaf were frailer than those receiving TCE. Hypogammaglobulinemia was more profound with TCE and the per patient risk of infections was higher. IVIg use was substantially higher for TCE than Belamaf. Blood viral PCR monitoring for TCE patients only detected symptomatic reactivations in 2 out of 13 patients suggesting this was an infrequent event.

An engineered immunocytokine with collagen affinity improves the tumor bioavailability, tolerability, and therapeutic efficacy of IL-2

The clinical utility of human interleukin-2 (hIL-2) is limited by its short serum half-life, preferential activation of regulatory T (TReg) over immune effector cells, and dose-limiting toxicities. We previously engineered F10 immunocytokine (IC), an intramolecularly assembled cytokine/antibody fusion protein that linked hIL-2 to an anti-IL-2 antibody (denoted F10) that extended IL-2 half-life and augmented the immune effector to TReg ratio. Here, we leveraged molecular engineering to improve the anti-tumor therapeutic efficacy and tolerability of F10 IC by developing an iteration, denoted F10 IC-CBD (collagen binding domain), designed for intratumoral administration and in situ retention based on collagen affinity. F10 IC-CBD retained IL-2 bioactivity exclusively in the tumor and eliminated IL-2-associated toxicities. Furthermore, F10 IC exhibited potent single-agent therapeutic efficacy and synergy with systemic immune checkpoint blockade and elicited an abscopal response in mouse tumors models. This engineered fusion protein presents a prototype for the design of intratumoral therapies.

Targeting DNA Repair and Survival Signaling in Diffuse Intrinsic Pontine Gliomas to Prevent Tumor Recurrence.

Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

A landscape of response to drug combinations in non-small cell lung cancer

Combination of anti-cancer drugs is broadly seen as way to overcome the often-limited efficacy of single agents. The design and testing of combinations are however very challenging. Here we present a uniquely large dataset screening over 5000 targeted agent combinations across 81 non-small cell lung cancer cell lines. Our analysis reveals a profound heterogeneity of response across the tumor models. Notably, combinations very rarely result in a strong gain in efficacy over the range of response observable with single agents. Importantly, gain of activity over single agents is more often seen when co-targeting functionally proximal genes, offering a strategy for designing more efficient combinations. Because combinatorial effect is strongly context specific, tumor specificity should be achievable. The resource provided, together with an additional validation screen sheds light on major challenges and opportunities in building efficacious combinations against cancer and provides an opportunity for training computational models for synergy prediction.