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Supplementary Materials and Methods Supplementary References Tables S1 to S3 and legends Figures S1 to S4 and legends

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC.Significance:These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Patients with brain tumors and metastases have poor prognosis and overall survival rates despite the advancements in neurosurgery, radiotherapy, and chemotherapy. Genomic alterations in HER2 are present in brain metastases of breast cancer (BC; ~20%) and non-small cell lung cancer (NSCLC; 13-20%), and EGFR alterations occur frequently in glioblastoma multiforme (GBM; >50%). Despite the advancements in standard of care options, optimal treatment management for these patients remains an unmet medical need. Recent evidence suggests activity of systemic therapy for immune and targeted therapies in the brain, including agents targeting HER2/EGFR. HER2-targeted tyrosine kinase inhibitors lapatinib, neratinib, and tucatinib and the HER2-targeted antibodies trastuzumab and pertuzumab, in combination with chemotherapy, have been shown to improve survival of patients with HER2 overexpressing BC in the presence of brain metastases. The limited penetration of these compounds into the CNS, however, limits their efficacy. TAS2940 is an irreversible pan-ErbB inhibitor with greater brain-penetrability than poziotinib, tucatinib, and neratinib. Here, we demonstrate that TAS2940 induces downregulation of phosphorylated HER2/EGFR, reduces tumor burden, and promotes a significant increase in survival in intracranial xenograft mouse models with HER2-amplification (BC), HER2-Exon20 insertion mutation (NSCLC), and EGFR-amplification (GBM). These promising preclinical data highlight potential novel therapeutic strategies for patients with EGFR-aberrant GBM and brain metastases harboring HER2/EGFR alterations, and may help support the advancement of the ongoing first-in-human clinical trial (NCT04982926) for TAS2940 in solid tumors with EGFR and/or HER2 alterations. Citation Format: Jing Han, Mikhila Mahendra, Poojabahen Gandhi, Joseph R. Daniele, Caroline C. Carrillo, Benjamin J. Bivona, Ningping Feng, John V. Heymach, Funda Meric-Bernstam, Kei Oguchi, Shinji Mizuarai, Timothy P. Heffernan, Christopher P. Vellano, Joseph R. Marszalek. TAS2940 inhibits intracranial tumor growth and prolongs survival in HER2-aberrant and EGFR-amplified patient-derived xenograft models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4017.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non–small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. Significance: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult cancers with an average 5-year survival rate of less than 10% due in part to the limited number of effective therapies. Activation of TRAILR2 (Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand Receptor 2) has emerged as an important therapeutic concept in cancer treatment. Traditional TRAILR2 agonists have had limited clinical success due to lack of efficacy or, importantly, severe hepatotoxicity. Here we present anti-tumor activity in preclinical PDAC models for BI 905711, a first-in-class tetravalent bispecific antibody specifically designed to overcome the disadvantages of previous strategies targeting TRAILR2. BI 905711 serves as a uniquely specific, and liver-sparing therapeutic by targeting tumors that co-express TRAILR2 and another cell surface protein CDH17, which has ~40% prevalence in PDAC and is not expressed in normal liver. Working from a large cohort of molecularly characterized PDAC PDX models, we provide the first preclinical evidence of BI 905711 exhibiting robust anti-tumor activity in difficult to treat PDAC PDX models. Anti-tumor efficacy in responding models correlated with strong induction of Caspases 3/7 and 8 activation in tumors 24 hours after a single dose of BI 905711, and was associated with the presence and expression levels of TRAILR2 and CDH17 proteins. Evaluation of models with differential TRAILR2 and CDH17 expression profiles helped define the expression threshold for each target that is associated with response, upon which clinical assay development is in process for future patient stratification. Additionally, response was also associated with PDAC molecular subtypes utilizing a novel proprietary gene co-expression network developed from a curated cohort of PDAC PDX tumors. Responders to BI 905711 were identified primarily within the classical and quasi-basal/hybrid subtypes when TRAILR2 was adequately co-expressed. This correlates with an enrichment pattern of CDH17 gene expression that is mostly within the classical gene cluster and strongly anti-correlated with basal-like cluster enrichment. Robust preclinical anti-tumor activity of BI 905711 in TRAILR2 and CDH17-expressing PDAC PDX models, along with this antibody's potential for a favorable safety profile, has justified the enrollment of pancreatic cancer patients in the ongoing BI 905711 FIH Phase I clinical trial (NCT04137289). Citation Format: Jing Han, Annette A. Machado, Mikhila Mahendra, Joseph R. Daniele, Christopher A. Bristow, Justin Kwang-Lay Huang, Alessandro Carugo, Robert A. Mullinax, Benjamin J. Bivona, Ningping Feng, Poojabahen Gandhi, Norbert Schweifer, Paolo Maria Chetta, Juan Manuel Garcia-Martinez, Frank Hilberg, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek. BI 905711 selectively induces apoptosis and anti-tumor response in TRAILR2/CDH17- expressing pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 985.

Chymase released from mast cells produces pro‐fibrotic, inflammatory, and vasoconstrictor agents. Studies were performed to test the hypothesis that chronic chymase inhibition provides a renal protective effect in type 2 diabetes. Diabetic (db/db) and control mice (db/m) were chronically infused with a chymase‐specific inhibitor or vehicle for 8 weeks. Baseline urinary albumin excretion (UalbV) averaged 42 ± 3 and 442 ± 32 microg/d in control (n = 22) and diabetic mice (n = 27), respectively (p, The most significant finding of the present study is the identification that chronic inhibition of chymase in the db/db type II diabetic mouse results in renoprotection as reflected by a significant attenuation of urinary albumin excretion and mesangial expansion/glomerular injury. These studies suggest that chymase inhibition may represent a novel therapeutic target in cardiovascular and renal disease.

We have previously reported significant increases in neuronal nitric oxide synthase (NOS) immunostaining in renal arterioles of angiotensin type 1A receptor (AT1A) knockout mice, and in arterioles and macula densa cells of AT1A/AT1B knockout mice. The contribution of nitric oxide derived from endothelial and macula densa cells in the maintenance of afferent arteriolar tone and acetylcholine-induced vasodilation was functionally determined in kidneys of wild-type, AT1A, and AT1A/AT1B knockout mice. Acetylcholine-induced changes in arteriolar diameters of in vitro blood-perfused juxtamedullary nephrons were measured during control conditions, in the presence of the nonspecific NOS inhibitor, Nω-nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N5-(1-imino-3-butenyl)-l-ornithine (VNIO). Acetylcholine (0.1 mM) produced a significant vasoconstriction in afferent arterioles of AT1A/AT1B mice (−10.9 ± 5.1%) and no changes in afferent arteriolar diameters of AT1A knockout mice. NLA (0.01–1 mM) or VNIO (0.01–1 μM) induced significant dose-dependent vasoconstrictions (−19.8 ± 4.0% 1 mM NLA; −7.8 ± 3.5% 1 μM VNIO) in afferent arterioles of kidneys of wild-type mice. VNIO had no effect on afferent arteriole diameters of AT1A knockout or AT1A/AT1B knockout mice, suggesting nonfunctional neuronal nitric oxide synthase. These data indicate that acetylcholine produces a significant renal afferent arteriole vasodilation independently of nitric oxide synthases in wild-type mice. AT1A receptors are essential for the manifestation of renal afferent arteriole responses to neuronal nitric oxide synthase-mediated nitric oxide release.

Angiotensin type 1A (AT1A) and 1B (AT1B) receptor deletion (AT1DKO) results in renal microvascular disease, tubulointerstitial injury, and reduced blood pressure. To test the hypothesis that renal preglomerular responses to angiotensin (ANG) II are mediated by AT1Aand AT1Breceptors, experiments were performed in AT1DKO mice using the in vitro blood perfused juxtamedullary nephron technique. Kidneys were harvested from AT1DKO and wild-type (WT) mice and bathed with ANG II (1–100 nM), norepinephrine (NE; 100–1,000 nM), or acetylcholine (ACh; 10 μM). Baseline diameters of afferent (19.5 ± 0.7 and 13.9 ± 0.7 μm, n = 17 and 16) and efferent (15.5 ± 2.1 and 10.8 ± 1.0 μm, n = 4 and 7) arterioles of AT1DKO were significantly larger than WT. Afferent and efferent arteriolar responses to ANG II, 100, and 300 nM NE were absent in AT1DKO; although significant constriction to 1 μM NE was observed (−17 ± 5 and −23 ± 6%, respectively). Afferent arterioles of WT mice dilated significantly in response to ACh (15.1 ± 0.6 to 17.0 ± 1.2 μm, n = 6); however, arterioles from AT1DKO tended to contract (19.9 ± 1.2 to 17.8 ± 1.6 μm; n = 6, P = 0.06). In summary, loss of ANG II-induced contraction, reduced vasoconstriction to NE, and endothelial cell dysfunction contribute to the renal vascular phenotype of AT1DKO mice. We conclude that ANG II signaling via the AT1receptor plays a pivotal role in basal renal microvascular tone and effectiveness to respond to vasoconstrictor and vasodilator agonists.

Background Our laboratory has previously demonstrated the importance of a cytoskeletal‐based survival signaling pathway using in vitro models of ischemia/reperfusion ( IR ). However, the importance of this pathway in mediating stress‐elicited survival signaling in vivo is unknown. Methods and Results The essential cytoskeletal signaling pathway member focal adhesion kinase ( FAK ) was selectively deleted in adult cardiac myocytes using a tamoxifen‐inducible Cre‐Lox system (α‐ MHC ‐MerCreMer). Polymerase chain reaction ( PCR ) and W estern blot were performed to confirm FAK knockout ( KO ). All mice were subjected to a 40‐minute coronary occlusion followed by 24 hours of reperfusion. Ischemic preconditioning ( IP ) was performed using a standard protocol. Control groups included wild‐type ( WT ) and tamoxifen‐treated α‐ MHC ‐MerCreMer +/− /FAK WT/WT (experimental control) mice. Infarct size was expressed as a percentage of the risk region. In WT mice IP significantly enhanced the expression of activated/phosphorylated FAK by 36.3% compared to WT mice subjected to a sham experimental protocol ( P ≤0.05; n=6 hearts [sham], n=4 hearts [ IP ]). IP significantly reduced infarct size in both WT and experimental control mice (43.7% versus 19.8%; P ≤0.001; 44.7% versus 17.5%; P ≤0.001, respectively). No difference in infarct size was observed between preconditioned FAK KO and nonpreconditioned controls (37.1% versus 43.7% versus 44.7%; FAK KO versus WT versus experimental control; P = NS ). IP elicited a 67.2%/88.8% increase in activated phosphatidylinositol‐3‐kinase ( PI 3K) p85/activated Akt expression in WT mice, but failed to enhance the expression of either in preconditioned FAK KO mice. Conclusions Our results indicate that FAK is an essential mediator of IP ‐elicited cardioprotection and provide further support for the hypothesis that cytoskeletal‐based signaling is an important component of stress‐elicited survival signaling.

Diabetic nephropathy (DN) is associated with enhanced renal, plasma, and urinary endothelin (ET)-1 levels. Chymase cleaves Big ET-1 (1-38) to ET-1 (1-31), which is further cleaved by neutral endopeptidase to ET-1 (1-21). The current study tested the hypothesis that afferent arterioles (AA) of diabetic kidneys exhibit enhanced vasoconstrictor responses to chymase-dependent intrarenal ET formation compared to control kidneys.In situ juxtamedullary AA vasoconstrictor responses to the intrarenal conversion of Big ET-1 (1-38) to ET-1 (1-21) were performed in the absence and presence of chymase inhibition in type 2 diabetic db/db and control db/m mice studied under in vitro experimental conditions.AA vasoconstrictor responses to Big ET-1 (1-38) were significantly enhanced in diabetic compared to control kidneys. In the presence of chymase inhibition (JNJ-18054478), AA vasoconstrictor responses of diabetic kidneys to Big ET-1 (1-38) were significantly less than the responses of control kidneys. AA diameters decreased similarly to ET-1 (1-21) in diabetic and control kidneys.AA responses to the intrarenal conversion of Big ET-1 (1-38) to ET-1 in the absence of chymase enzymatic activity were significantly reduced in kidneys of diabetic compared to control mice, while the magnitude of the vasoconstriction to ET-1 (1-21) was not different. These data suggest that AA vasoconstriction produced by the chymase-dependent pathway is significantly greater in diabetic compared to control kidneys. We propose that intrarenal chymase-dependent ET-1 production contributes to the decline in function and progression to end-stage renal disease in patients with type 2 diabetes.

Diabetic nephropathy is a major cause of end-stage renal disease worldwide. The current studies were performed to determine the later stages of the progression of renal disease in type II diabetic mice (BKS; db/db). Methodology was developed for determining glomerular filtration rate (GFR) in conscious, chronically instrumented mice using continuous intravenous infusion of FITC-labeled inulin to achieve a steady-state plasma inulin concentration. Obese diabetic mice exhibited increased GFR compared with control mice. GFR averaged 0.313 ± 0.018 and 0.278 ± 0.007 ml/min in 18-wk-old obese diabetic ( n = 11) and control ( n = 13) mice, respectively ( P < 0.05). In 28-wk-old obese diabetic ( n = 10) and control ( n = 15) mice, GFR averaged 0.348 ± 0.030 and 0.279 ± 0.009 ml/min, respectively ( P < 0.05). GFR expressed per gram BW was significantly reduced in 18- and 28-wk-old obese diabetic compared with control mice (5.9 ± 0.3 vs. 9.0 ± 0.3; 6.6 ± 0.6 vs. 7.8 ± 0.3 μl·min−1·g body wt−1), respectively ( P < 0.05). However, older nonobese type II diabetic mice had significantly reduced GFR (0.179 ± 0.023 ml/min; n = 6) and elevated urinary albumin excretion (811 ± 127 μg/day) compared with obese diabetic and control mice (514 ± 54, 171 ± 18 μg/day), which are consistent with the advanced stages of renal disease. These studies suggest that hyperfiltration contributes to the progression of renal disease in type II diabetic mice.

Combination therapy of angiotensin-converting enzyme (ACE) inhibition and AT1receptor blockade has been shown to provide greater renoprotection than ACE inhibitor alone in human diabetic nephropathy, suggesting that ACE-independent pathways for ANG II formation are of major significance in disease progression. Studies were performed to determine the magnitude of intrarenal ACE-independent formation of ANG II in type II diabetes. Although renal cortical ACE protein activity [2.1 ± 0.8 vs. 9.2 ± 2.1 arbitrary fluorescence units (AFU)·mg−1·min−1] and intensity of immunohistochemical staining were significantly reduced and ACE2 protein activity (16.7 ± 3.2 vs. 7.2 ± 2.4 AFU·mg−1·min−1) and intensity elevated, kidney ANG I (113 ± 24 vs. 110 ± 45 fmol/g) and ANG II (1,017 ± 165 vs. 788 ± 99 fmol/g) levels were not different between diabetic and control mice. Afferent arteriole vasoconstriction due to conversion of ANG I to ANG II was similar in magnitude in kidneys of diabetic (−28 ± 3% at 1 μM) and control (−23 ± 3% at 1 μM) mice; a response completely inhibited by AT1receptor blockade. In control kidneys, afferent arteriole vasoconstriction produced by ANG I was significantly attenuated by ACE inhibition, but not by serine protease inhibition. In contrast, afferent arteriole vasoconstriction produced by intrarenal conversion of ANG I to ANG II was significantly attenuated by serine protease inhibition, but not by ACE inhibition in diabetic kidneys. In conclusion, there is a switch from ACE-dependent to serine protease-dependent ANG II formation in the type II diabetic kidney. Pharmacological targeting of these serine protease-dependent pathways may provide further protection from diabetic renal vascular disease.

The db/ db mouse is a genetic model of type 2 diabetes that exhibits progressive renal disease. Obesity, hyperglycemia, and albuminuria (822 ± 365 vs. 28 ± 8 μg/day) are evident in 18-wk-old db/ db compared with db/ m (lean littermate control) mice. Our goal was to determine the blood pressure (BP) phenotype of the db/ db mouse. Mean arterial BP measured in conscious mice by radiotelemetry was not different between db/ db ( n = 9) and db/ m ( n = 12) mice, averaging 113 ± 3 and 112 ± 2 mmHg, respectively. The circadian BP profile of db/ db mice was shifted to the left and exhibited a significant reduction in amplitude compared with db/ m mice. Heart rate (487 ± 9 vs. 542 ± 7 beats/min; P < 0.05) and locomotor activity were significantly reduced in db/ db compared with db/ m mice. We tested the hypothesis that intact afferent arteriole (AA) responsiveness to increases in renal artery pressure (RAP) and angiotensin (ANG) II sensitivity contributes to normal BP in this diabetic model. AA diameters of in vitro blood-perfused juxtamedullary nephrons of db/ db mice (15.7 ± 0.5 μm; n = 38) were significantly larger than those of db/ m mice (12.5 ± 0.4 μm; n = 37). AA responses to increases in RAP and ANG II were not different between kidneys of db/ db and db/ m mice. Significant AA vasoconstriction to 1 nM ANG II was observed in kidneys of db/ db mice (−11 ± 4%), while 10 nM ANG II decreased AA diameter in both groups [ db/ db, −20 ± 4%, ( n = 12); db/ m, −26 ± 4% ( n = 12)]. In summary, AA responses to increases in renal perfusion pressure and ANG II remain intact in db/ db mice. Diabetic renal disease occurs in db/ db mice independently of elevated BP.

Angiotensin (ANG) type 1A (AT1A) receptor-null (AT1A−/−) mice exhibit reduced afferent arteriolar (AA) constrictor responses to ANG II compared with wild-type (WT) mice, whereas efferent arteriolar (EA) responses are absent (Harrison-Bernard LM, Cook AK, Oliverio MI, and Coffman TM. Am J Physiol Renal Physiol 284: F538–F545, 2003). In the present study, the renal arteriolar constrictor responses to norepinephrine (NE) and/or ANG II were determined in blood-perfused juxtamedullary nephrons from kidneys of AT1A−/−, AT1B receptor-null (AT1B−/−), and WT mice. Baseline AA diameter in AT1A−/− mice was not different from that in WT mice (13.1 ± 0.9 and 12.6 ± 0.9 μm, n = 7 and 8, respectively); however, EA diameters were significantly larger (17.3 ± 1.4 vs. 11.7 ± 0.4 μm, n = 10 and 8) in AT1A−/− than in WT mice. Constriction of AA (−40 ± 8 and −51 ± 6% at 1 μM NE) and EA (−29 ± 6 and −38 ± 3% at 1 μM NE) in response to 0.1–1 μM NE was similar in AT1A−/− and WT mice. Baseline diameters of AA (13.5 ± 0.7 and 14.2 ± 0.9 μm, n = 9 and 10) and EA (15.4 ± 1.0 and 15.0 ± 0.7 μm, n = 11 and 9) and ANG II (0.1–10 nM) constrictor responses of AA (−25 ± 4 and −31 ± 5% at 10 nM) and EA (−32 ± 6 and −35 ± 7% at 10 nM) were similar in AT1B−/− and WT mice, respectively. ANG II-induced constrictions were eliminated by AT1 receptor blockade with 4 μM candesartan. Taken together, our data demonstrate that AA and EA responses to NE are unaltered in the absence of AT1A receptors, and ANG II responses remain intact in the absence of AT1B receptors. Therefore, we conclude that AT1A and AT1B receptors are functionally expressed on the AA, whereas the EA exclusively expresses the AT1A receptor.