Potential Pharmacodynamic Biomarkers Research Articles

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pathogenic mutations in LRRK2 cause Parkinson’s disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

S100A8/A9 predicts response to PIM kinase and PD-1/PD-L1 inhibition in triple-negative breast cancer mouse models

BackgroundUnderstanding why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well remains a challenge. This study aims to understand the potential underlying mechanisms distinguishing early-stage TNBC tumors that respond to clinical intervention from non-responders, as well as to identify clinically viable therapeutic strategies, specifically for TNBC patients who may not benefit from existing therapies.MethodsWe conducted retrospective bioinformatics analysis of historical gene expression datasets to identify a group of genes whose expression levels in early-stage tumors predict poor clinical outcomes in TNBC. In vitro small-molecule screening, genetic manipulation, and drug treatment in syngeneic mouse models of TNBC were utilized to investigate potential therapeutic strategies and elucidate mechanisms of drug action.ResultsOur bioinformatics analysis reveals a robust association between increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors and subsequent disease progression in TNBC. A targeted small-molecule screen identifies PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types, including TNBC and immunosuppressive myeloid cells. Combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses, especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Notably, serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC.ConclusionsOur data propose S100A8/A9 as a potential predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC. This work encourages the development of S100A8/A9-based liquid biopsy tests for treatment guidance.

Targeting EIF4A triggers an interferon response to synergize with chemotherapy and suppress triple-negative breast cancer.

Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A by Zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages towards an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that Zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon response uniformly across models. The induction of an interferon response is partially due to the inhibition of Sox4 translation by Zotatifin. A similar induction of interferon-stimulated genes was observed in breast cancer patient biopsies following Zotatifin treatment. Surprisingly, Zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened interferon response resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for Zotatifin, and provide a rationale for new combination regimens comprising Zotatifin and chemotherapy or immunotherapy as treatments for TNBC.

Antihypertensive activity of different components of Veratrum alkaloids through metabonomic data analysis

BackgroundHypertension is a serious global public health issue. Blood pressure (BP) is still not effectively controlled in about 20 – 30% of hypertensive patients. Therefore, it is imperative to develop new treatments for hypertension. Veratrum alkaloids were once used for the clinical treatment of hypertension, the mechanism of which is still unclear. It was gradually phased out due to adverse reactions. PurposeThis study aimed to investigate the short-term and long-term hypotensive profiles of different components of Veratrum alkaloids in spontaneously hypertensive rats (SHRs) to unveil their mechanisms of action. ResultsTotal Veratrum alkaloid (V), component A (A), and veratramine (M) quickly decreased BP within 30 min of treatment, reduced renal and cardiovascular damage, and improved relevant biochemical indicators (nitric oxide [NO], endothelin-1 [ET-1], angiotensin II [Ang II)], noradrenaline [NE], etc) in SHRs to delay stroke occurrence. Thereinto, A exhibited excellent protective effects in cardiovascular disease. The metabolomic profiles of SHRs treated with V, A, and M were significantly different from those of SHRs treated with vehicle. Thirteen metabolites were identified as potential pharmacodynamic biomarkers. Through Kyoto Encyclopedia of Genes and Genomes analysis, V, A, and M-induced hypotension was mainly related to alterations in nicotinate and nicotinamide metabolism, GABAergic synapses, linoleic acid metabolism, ketone body synthesis and degradation, arginine and proline metabolism, and urea cycle, of which nicotinate and nicotinamide metabolism was the key metabolic pathway to relieve hypertension. ConclusionThis work shows that A is an effective and promising antihypertensive agent for hypertension treatment to reduce BP and hypertensive target organ damage, which is mainly mediated through modulating nicotinate and nicotinamide metabolism, RAS, and NO–ET homeostasis.

Abstract 2119: Intravenous vitamin C therapy for cisplatin ineligible bladder cancer patients (CI-MIBC): Early investigation into potential pharmacodynamic biomarkers

Abstract Neoadjuvant cisplatin based chemotherapy is considered standard of care for patients with locally advanced bladder cancer (BCa). However, upwards of 50% of patients are ineligible due to poor performance status, underlying kidney disease, peripheral neuropathy, hearing loss and/or cardiac disease. There are currently no accepted alternative regimens with significant activity, and many cisplatin ineligible patients usually proceed directly to surgery. Prior research in other cancers has indicated intravenous vitamin C (IVC), or ascorbate, may serve as a beneficial adjuvant to less toxic therapeutic regimens. We initiated a single arm, Simon 2-stage, Window of Opportunity trial using high dose IVC with single cycle gemcitabine/carboplatin (GCa) (NCT04046094). The primary outcome was pathologic downstaging. Twelve patients with newly diagnosed CI-MIBC were enrolled. Patients received single cycle GCa and IVC titrated to 350-400 mg/dL for ~21 days followed by cystectomy after ~4-6 weeks. As reported, pathological downstaging (<ypT2N0Mx) was present in 4 (33%) of patients. Three of the four patients had a complete response (ypT0NoMx) including one patient with plasmacytoid variant histology. Treatment was well tolerated with no attributable AE/SAEs. Given the discrepancy in response levels, we began identifying potential markers of pharmacodynamic efficacy to inform future trials. Novel data evaluating Ki67+ cells by immunohistochemistry indicate widespread proliferation in non-responders to IVC/GCa, whereas responding patients had visibly fewer positive cells, suggesting a direct effect on cell death and proliferation. TUNEL staining was present in all samples, although often minimally, and thus TUNEL may not yield enough signal to reliably make determinations about treatment outcomes. Urinary cytokine values were assessed using multiplex ELISA and reported as pg/mL urine. Multiple cytokines including IL-1RA, CCL2, IL-8, CXCL10 were present at markedly high concentrations in patients with BCa. Cytokine levels generally increased between diagnostic resection and radical cystectomy indicating potential immune activation by the IVC/GCa treatment. Cytokine levels generally fell between radical cystectomy and follow-up samples suggesting resolution of inflammation after cystectomy. In conclusion, IVC/GCa may be a viable alternative neoadjuvant treatment strategy for patients with BCa that are ineligible for cisplatin-based chemotherapy. Identifying biomarkers of response remains a major area of interest to refine study designs and identify patients most likely to benefit from therapy. Citation Format: Benjamin L. Woolbright, Ganeshkumar Rajendran, Erika Abbott, Zeb Zacharias, Jon Houtman, Rahul Parikh, Qi Chen, Ameer Hamza, Elizabeth Wulff-Burchfield, Jeffrey M. Holzbeierlein, Jeanne Drisko, Michael D. Henry, John A. Taylor. Intravenous vitamin C therapy for cisplatin ineligible bladder cancer patients (CI-MIBC): Early investigation into potential pharmacodynamic biomarkers [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 2119.

Abstract 6129: The proapoptotic peptide PEP-010 is efficient on several models of different tumor origins and it can be monitored by pharmacodynamic biomarker candidates in clinical practice

Abstract In cancer cells, the proapoptotic proteins caspase-9 and PP2A interaction prevents them to play their role in apoptosis. PEP-010, a drug candidate developed by PEP-Therapy, a French Biotech company, is a proapoptotic peptide targeting and disrupting this interaction leading to restoration of the apoptotic cascade. PEP-010 is an innovative bifunctional peptide. It penetrates into cells thanks to its cell penetrating part and specifically disrupts the caspase-9/PP2A interaction thanks to its interfering part. PEP-010 is currently in Phase I a/b multicenter clinical trial for the treatment of advanced solid tumors.To the aim of screening different potential therapeutic targets, to investigate the molecular mechanism-of-action of PEP-010 and to identify potential pharmacodynamic biomarkers, we have tested PEP-010 efficacy on several cell models of different tumor origins. By the Annexin V/Propidium Iodide staining and flow cytometry analysis, we found that PEP-010 induces apoptosis in most of the tested models. One possible mechanism-of-action is an involvement of the PP2A which, once released by PEP-010, could regulate expression and/or activity of different intracellular factors leading to caspases activation and cell death.The identification of the major molecular features involved in PEP-010 mechanism-of-action, were instrumental to identify different pharmacodynamic biomarker candidates (e.g. active caspase-3). Hence, such biomarkers could be useful in clinical practice to monitor the effect of PEP-010 at molecular level. We have compared specific features related to the mechanism-of-action of PEP-010 in sensitive (MDA-MB-231, IGROV1) and not sensitive cell models of different tumor origins and in tissue sections derived from Patient-Derived Xenografts models of breast cancer treated or not with PEP-010. Widely used techniques as immunofluorescent staining and immunohistochemistry were employed, making these results easily transferable in clinical routine.Taken together, our pre-clinical data showed the potential of PEP-010 as an anti-cancer peptide on a wide variety of malignancies and enabled the identification of pharmacodynamic biomarker candidates, important to ease the clinical development. Citation Format: Rayan Farhat, Laura Dadon, F Nemati, A Rebollo, D Decaudin, Joelle Wiels, Catherine Brenner, Diego Germini. The proapoptotic peptide PEP-010 is efficient on several models of different tumor origins and it can be monitored by pharmacodynamic biomarker candidates in clinical practice. [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 6129.

Combination of naproxen and a chemically-stable eicosapentaenoic acid analog provide additive tumor protection in Pirc rats.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Patients with the genetic disorder Familial Adenomatous Polyposis (FAP) develop hundreds to thousands of polyps that unless removed by prophylactic colectomy will progress to CRC at an early age. Nonsteroidal anti-inflammatory drugs (NSAIDs) and the ω-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA), have been evaluated for their chemopreventive potential in delaying CRC onset in high-risk patients. In our study, we determined whether the NSAID, naproxen, alone or in combination with a chemically-stable EPA analog (TP-252), affects tumor formation in the ApcPirc rat model. When compared to control diet, animals fed naproxen or HD TP-252 had 66% and 82% fewer tumors, respectively. However, animals fed a combination of naproxen and HD TP-252, exhibited a 95% reduction in tumor formation and a 98% reduction in tumor volume, respectively. To elucidate potential mechanisms of tumor protection, a comprehensive, targeted lipidomic analysis was performed on colonic mucosa to determine changes in eicosanoid metabolism. Animals receiving TP-252 alone or in combination with naproxen had significantly reduced mucosal levels of proinflammatory ω-6 eicosanoids (PGE2 , 5-HETE and 14,15-DiHETrE), along with a simultaneous increase in anti-inflammatory EPA-derived ω-3 eicosanoids. A comprehensive lipidomic analysis also uncovered several potential pharmacodynamic (PD) lipid biomarkers, including resolvin E2, 9-HEPE, 12-HEPE and 18-HEPE, that were significantly correlated with tumor protection. Further studies with this drug combination should be focused on dose optimization and the role of EPA-derived lipid mediators in CRC initiation and progression.

Longitudinal evaluation of serum microRNAs as biomarkers for neuroblastoma burden and therapeutic p53 reactivation.

Accurate assessment of treatment response and residual disease is indispensable for the evaluation of cancer treatment efficacy. However, performing tissue biopsies for longitudinal follow-up poses a major challenge in the management of solid tumours like neuroblastoma. In the present study, we evaluated whether circulating miRNAs are suitable to monitor neuroblastoma tumour burden and whether treatment-induced changes of miRNA abundance in the tumour are detectable in serum. We performed small RNA sequencing on longitudinally collected serum samples from mice carrying orthotopic neuroblastoma xenografts that were exposed to treatment with idasanutlin or temsirolimus. We identified 57 serum miRNAs to be differentially expressed upon xenograft tumour manifestation, out of which 21 were also found specifically expressed in the serum of human high-risk neuroblastoma patients. The murine serum levels of these 57 miRNAs correlated with tumour tissue expression and tumour volume, suggesting potential utility for monitoring tumour burden. In addition, we describe serum miRNAs that dynamically respond to p53 activation following treatment of engrafted mice with idasanutlin. We identified idasanutlin-induced serum miRNA expression changes upon one day and 11 days of treatment. By limiting to miRNAs with a tumour-related induction, we put forward hsa-miR-34a-5p as a potential pharmacodynamic biomarker of p53 activation in serum.

P188: Urine glucose tetrasaccharide as a potential pharmacodynamic biomarker for the development of new treatments for glycogen storage disease type III*

P188: Urine glucose tetrasaccharide as a potential pharmacodynamic biomarker for the development of new treatments for glycogen storage disease type III*

Abstract A005: Identification of lipid pharmacodynamic biomarkers in FAP model

Abstract Familial Adenomatous Polyposis (FAP) is a genetic disorder characterized by the development of numerous polyps in the colon, that unless removed by colectomy, inevitably progress to colorectal cancer (CRC). Although there is currently no FDA-approved treatment to delay colectomy, a common target for chemoprevention is cyclooxygenase-2 (COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs) and omega-3 fatty acids are known to alter COX metabolism and affect downstream eicosanoids involved in CRC progression. In this study, we examined the chemopreventive efficacy of the NSAID naproxen alone, or in combination with TP-252, a novel, chemically-stable analogue of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in the intestinal tumorigenesis Apcam1137 PIRC rat model. Over the course of this 20-week study, rats were fed a modified AIN-93G diet supplemented with 200 ppm naproxen, alone or in combination with TP-252 (1.5%, or 3% by weight). Rats treated with 200 ppm naproxen had 66% (p < 0.0001) fewer colon tumors than controls (AIN-93G) and an 81% (p < 0.0001) reduction in tumor size. However, when Naproxen was combined with high-dose TP-252 (3%), tumor number and size were reduced by 95% (p < 0.0001) and 98% (p < 0.0001) respectively, when compared to controls. Since the primary mechanisms of action for naproxen and EPA target COX metabolic activity, we performed a comprehensive lipidomic analysis on colon tissue and plasma to determine lipidomic changes associated with drug treatment. Increased formation of several EPA-derived eicosanoids, including 9-HEPE, 12-HEPE, 18-HEPE, and resolvin E2, whose mucosal levels correlated with both reduced tumor number and volume, were observed. These metabolites were also concomitantly elevated in the plasma of treated animals, representing potential pharmacodynamic (PD) biomarkers of CRC prevention. Future studies will decipher potential mechanisms that underlie these EPA-derived PD biomarkers and their role in tumor suppression. (Supported by the NCI Prevent Program 75N91019D00019, Task Order 75N91019F00132). Citation Format: Ryan Beach, Altaf Mohammed, Daniel W. Rosenberg. Identification of lipid pharmacodynamic biomarkers in FAP model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A005.

Abstract 5448: Synergistic antitumor effect of ONC201, radiotherapy and temozolomide in glioblastoma mouse orthotopic models

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults with a median survival of just over a year and a two-year survival of about 30%. Only 10% of people survive five years of this grade IV cancer. Standard of care therapy comprises maximal safe surgical resection followed by adjuvant alkylating agent temozolomide (TMZ) and radiotherapy conferring a median survival time of 14.6 months compared with 12.1 months for GBM patients treated with radiotherapy alone. First-in-class small-molecule imipridone ONC201 has shown efficacy in glioblastoma models in vitro, in vivo, and ex vivo. It is also orally active and is administered once every week, a favorable dosing schedule for patients. We hypothesized that ONC201 may synergize with radiotherapy and Temozolomide in GBM in vitro and in vivo. We previously reported that ONC201 synergizes with radiotherapy and/or TMZ to inhibit cell viability and colony formation and induce ER stress and apoptosis in GBM cells. To pursue this promising combinational therapy in vivo, we established GBM mouse orthotopic models through intracranial injection of luciferase expressing GBM cells U-251-LUC with a KOPF model 940 small animal stereotaxic frame and a Stoelting Quintessential Stereotaxic Injector. Tumor formation was confirmed with bioluminescence imaging. Randomized treatment group mice have received weekly treatment of ONC201 (100 mg/kg p.o.) and/or radiotherapy (2 Gy local irradiation) and/or TMZ (20 mg/kg i.p.) for four weeks. We observed the triple combination of ONC201 and radiotherapy and TMZ significantly reduced tumor burden and prolonged survival compared to dual combinations in GBM mouse orthotopic model U-251-LUC. We are currently exploring the synergistic mechanisms of triple combination including TRAIL signaling pathway, ISR, dopamine receptors and mitochondrial ClpXP, and potential pharmacodynamic biomarkers from in vivo samples. Our study suggests that the investigational triple combination therapy of ONC201 and radiotherapy and TMZ is going to be an important option for patients with GBM. Citation Format: Lanlan Zhou, Jinxuan Laura Wu, Howard P. Safran, Wafik S. El-Deiry. Synergistic antitumor effect of ONC201, radiotherapy and temozolomide in glioblastoma mouse orthotopic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5448.

Efficacy of EGFR Inhibitors and NSAIDs Against Basal Bladder Cancers in a Rat Model: Daily vs. Weekly Dosing, Combining EGFR Inhibitors with Naproxen, and Effects on RNA Expression

BACKGROUND: There are few effective treatments specifically aimed at basal bladder cancer. OBJECTIVE: Female F344 rats administered N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) develop large invasive bladder cancers. We determined the efficacy of daily vs weekly dosing of EGFR inhibitors, determined the efficacy of naproxen combined with an EGFR inhibitor, and performed RNA analysis of bladder tumors treated for 5 days with EGFR inhibitors or NO-naproxen to identify pharmacodynamic biomarkers. METHODS: Erlotinib (6 mg/Kg BW daily or 21 or 42 mg/Kg BW weekly), lapatinib (25 or 75 mg/Kg BW daily or 263 or 525 mg/Kg BW weekly) and/or naproxen (30 mg/Kg BW daily) were administered to OH-BBN-treated rats beginning 2–12 weeks post OH-BBN. Rats were sacrificed 28 weeks after the final OH-BBN treatment to determine the effects of the EGFR inhibitors + naproxen on bladder weights and tumor development. In a separate study, rats were treated with OH-BBN. When palpable tumors developed, rats were treated with erlotinib, lapatinib, gefitinib, or the NSAID NO-naproxen for 5 days. RNA analysis was performed on the tumors. RESULTS: Daily or weekly dosing of erlotinib or lapatinib and daily dosing of naproxen reduced large tumor formation up to 70%, while combining daily lapatinib and naproxen reduced tumors 100%. RNA Analysis: All EGFR inhibitors strongly reduced cell proliferation and chromosome replication pathways, while NO-naproxen altered the G protein receptor, oxygen homeostasis and immune function pathways. CONCLUSIONS: While daily and weekly dosing with EGFR inhibitors and naproxen were effective, combining lapatinib and naproxen yielded no tumors. This might encourage its clinical use in an adjuvant setting with superficial basal tumors, and perhaps even in a more advanced setting. Furthermore, RNA analysis identified specific pathways that might be potential pharmacodynamic biomarkers in clinical trials.

Urinary metabonomics study of anti-depressive mechanisms of Millettia speciosa Champ on rats with chronic unpredictable mild stress-induced depression

As a traditional Chinese medicine (TCM), Millettia speciosa Champ (MSC), exerts a wide range of pharmacological activities. Our research group previously found that MSC has antidepressant effects, but the specific antidepressant mechanisms remain unclear. Therefore, in this study, urine metabolomics based on ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) combined with pharmacodynamics was used to explore the pathogenesis of depression and the antidepressant effects of MSC. The results showed that MSC treatment could significantly improve chronic unpredictable mild stress (CUMS)-induced depression. Urine metabolic showed that the profiles of the CUMS model group were significantly separated from the control group, while the drug-treated groups were closer to the control group, especially the MSC group treated with a 14 g/kg dose of MSC. Furthermore, 9 metabolites, including glutaric acid, L-isoleucine, L-Dopa, sebacic acid, 3-methylhistidine, allantoin, caprylic acid, tryptophol, and 2-phenylethanol glucuronide, were identified as potential biomarkers of depression. Metabolic pathway analysis showed that these potential biomarkers were mainly involved in valine, leucine, and isoleucine biosynthesis, aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine degradation, tyrosine metabolism, histidine metabolism, fatty acid biosynthesis, and pentose and glucuronate interconversions. Through Receiver operating characteristic (ROC) analysis and Pearson correlation analysis, the combination of L-isoleucine, sebacic acid, and allantoin, were further screened out as potential pharmacodynamic biomarkers associated with the efficacy of MSC. This study suggests that the integration of metabolomics with pharmacodynamics helps to further understand the pathogenesis of depression and provides novel insight into the efficacy of TCM.

Abstract 385: Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma

Abstract Background: IRAK4, a serine/threonine kinase, was identified as a therapeutic target in hematological malignancies. IRAK4-mediated activation of NF-kappaB could play an important role in NF-kappaB-regulated survival and chemoresistance of cancer cells. CA-4948, a first-in-class small molecule inhibitor of IRAK4, has been tested in Phase 1 clinical trial with evidence of clinical activity in Non-Hodgkin's Lymphoma (NHL) patients. To support the development of companion diagnostic for CA-4948, we developed an immunohistochemical (IHC) assay and explored expression of potential biomarkers of response to CA-4948 in tumor biopsy samples obtained from NHL patients. Materials and Methods: IHC assay was developed and used for analysis of NF-kappaB p-p50 and p-IRAK1 expression in tumor biopsy samples obtained from 14 patients with NHL. Patients were defined according to their clinical response to CA-4948 treatment: stable disease (SD), 6 cases and progressive disease (PD), 8 cases. Results: We found nuclear and/or cytoplasmic expression of NF-kappaB p-p50 in all 6 SD cases treated with 50 mg QD (2 cases, tumor regression), 50 mg BID (1 case), 200 mg BID (1 case, tumor regression) and 400 mg BID (2 cases). Expression of NF-kappaB p-p50 was not detected in 7 of 8 cases with PD including patients treated with 50 mg QD (1 case), 100 mg QD (1 case), 100 mg BID (3 cases), 200 mg BID (1 case) and 400 mg BID (1 case). We found statistically significant correlation between expression of NF-kappaB p-p50 in tumor biopsy and SD in NHL patients treated with CA-4948 (p<0.05). Analysis of NF-kappaB p-p50 expression in paired tumor biopsy samples (3 cases) collected before and after the treatment with CA-4948 revealed a significant downregulation of NF-kappaB p-p50 expression in tumors obtained from CA-4948-treated NHL patients. In support of our in vivo findings, our in vitro experiments demonstrated that expression of NF-kappaB p-p50 was depleted in 3D lymphoma organoids treated with a clinically relevant concentration of CA-4948. Our results support further development of NF-kappaB p-p50 as a potential predictive and pharmacodynamic biomarker of IRAK4 inhibitor CA-4948. We found that expression of p-IRAK1 in tumor biopsies was not correlated with clinical response. Conclusions: Although the cohort size is small, our findings suggest that expression of NF-kappaB p-p50 can serve as biomarker to predict SD in response to the treatment with IRAK4 inhibitor CA-4948 in NHL patients. NF-kappaB p-p50 selection strategy might be used in future clinical trials to identify NHL patients which are most likely to respond to CA-4948 in combination with chemotherapy or targeted therapeutics. Citation Format: Andrey Ugolkov, Maria Samson, Rosanna Hok, Reinhard von Roemeling, Robert Martell. Identification of NF-kappaB phospho-p50 as a predictive biomarker for IRAK4 inhibitor CA-4948 in patients with non-Hodgkin's lymphoma [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 385.

Abstract CT207: Phase 1 first-in-human study of ABBV-151 as monotherapy or in combination with budigalimab in patients with locally advanced or metastatic solid tumors

Abstract Background Glycoprotein-A repetitions predominant (GARP) regulates membrane-bound transforming growth factor β1 (TGFβ1), an immunosuppressive cytokine. ABBV-151 is a first-in-class monoclonal antibody (mAb) that binds to the GARP-TGFβ1 complex and blocks TGFβ1 release. Preclinical data demonstrated that targeting both GARP-TGFβ1 and programmed cell death protein 1 (PD-1) improved antitumor effects compared with anti-PD-1 alone. Combining ABBV-151 with the anti-PD-1 mAb budigalimab (ABBV-181) may enable a more effective antitumor immune response by reducing the immunosuppressive effect of TGFβ1. Trial design This is a multicenter phase 1, dose escalation and dose expansion study (NCT03821935) in patients (pts; ≥18 yr, Eastern Cooperative Oncology Group performance status 0-1) with locally advanced or metastatic solid tumors. The primary objective of dose escalation is to determine the recommended phase 2 dose (RP2D) of ABBV-151 as monotherapy or with budigalimab; dose expansion will assess the objective response rate of ABBV-151 ± budigalimab. Secondary/exploratory objectives include assessing preliminary efficacy, safety, tolerability, pharmacokinetics (PK), and evaluating potential pharmacodynamic and predictive biomarkers. Dose escalation of ABBV-151, guided by a Bayesian optimal interval design, will assess dose-limiting toxicities during the first 28-day cycle and will be utilized until the RP2D is defined. ABBV-151 + budigalimab (fixed dose) will start ≥2 dose levels below that proven safe for ABBV-151. Adverse events will be evaluated per National Cancer Institute Common Terminology Criteria v5.0. Response will be assessed using Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 and iRECIST every 8 weeks. PK of ABBV-151 will be characterized. Saturation of GARP-TGFβ1 on platelets and PD-1 on CD4 T cells will be determined. Modulation of cytokines, chemokines, lymphocyte activity, and gene expression will be assessed in blood, while gene signatures and protein markers will be explored in tumor tissues. Baseline tumor characteristics will be retrospectively related to response. Enrollment initiated Mar 2019, with 37 pts enrolled as of May 2020. Citation Format: John Powderly, Toshio Shimizu, Patricia LoRusso, Albiruni Razak, Kathy Miller, Arjun Balar, Jordi Bruix, Loren Michel, Martha Blaney, Xiaowen Guan, Susan Lacy, Satwant Lally, Stacie Lambert, Rachel Leibman, Gregory Vosganian, Talia Golan, Anthony Tolcher. Phase 1 first-in-human study of ABBV-151 as monotherapy or in combination with budigalimab in patients with locally advanced or metastatic solid tumors [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 CT207.

Identification of Acetylation, Immune Cell Alterations and Gene Signature As Dynamic Biomarkers of Response to Panobinostat Treatment in Multiple Myeloma Patients

Introduction and aims:Panobinostat is a pan-deacetylase inhibitor (DACi) that affects multiple oncogenic and immune-stimulating genes required for tumour cell growth and survival through acetylation of both histone and non-histone proteins. Evaluation of panobinostat as single agent consolidation in patients post-melphalan 200mg/m2 conditioned autologous stem cell transplant (ASCT) was performed in a Phase II, open label, single arm study, wherein newly diagnosed patients (n=35) with less than complete response following ASCT were enrolled. Eighteen patients (12 responders - R, 6 non-responders - NR) were subsequently selected for correlative analysis to identify dynamic biomarkers that could predict for response to treatment. Acetylation is the key downstream effect of treatment with DACi and was therefore assessed to determine its validity as a pharmacodynamic marker of response. The proportions of different immune subsets at specific time-points during treatment were also assessed to identify differences in the immune composition between R and NR. Gene transcription studies were performed to detect differentially regulated genes/pathways.MethodsFor all correlative studies, screening sample (Pre-ASCT), post - ASCT, end of cycle 3 (EOC3) and EOC6 were utilised. Peripheral blood mononuclear cells (PBMC) from post-ASCT, EOC3 and EOC6 were processed to determine acetylation status of lysine residues (K) in histone 2A - H2A (K5), H2B (K5), H3 (K9, K14, K18 and K56), H4 (K5, K8, K12 and K16) and tubulin in CD3+ (T-lymphocytes) utilising flow cytometry. Changes were measured using mean fluorescence intensity (MFI) of the geometric mean. The MFI from a sample was normalized to the respective control sample and expressed as a fold difference. PBMC from all 4 time points were also assessed for proportions of different subsets of T-lymphocytes (T-helper, cytotoxic T-cells and T-regs) and natural killer (NK) cells using flow cytometry. Whole bone marrow (BM) aspirates were collected in PAXgene blood RNA tubes and extracted RNA was subjected to 100 bp paired-end total RNA sequencing.Results and conclusion:Panobinostat-induced acetylation in T-lymphocytes was significantly higher at EOC3 (H2B K5, H3 K9, H3 K14, H3 K56, H4 K8, H4 K12, H4 K16 (p<0.01)) and EOC6 (all histones assessed, p<0.05) in R, with no differences in NR at EOC3 and EOC6 compared to post-ASCT, suggesting that acetylation could be utilised as a pharmacodynamic biomarker of response to panobinostat. Assessment of T-cell proportions in this annotated sample set indicated that the ratio of CD4/CD8 T-cells was significantly higher at EOC3 in R compared to NR (p<0.01), consistent with published literature, which demonstrates that the ratio of CD4/CD8 is significantly lower in patients with poor prognostic tumors. Furthermore, MFI of CD57 in cytotoxic cells was higher in EOC3 and EOC6 compared to post-ASCT in R while it was downregulated in NR (p<0.05), also suggesting an increased cytotoxic activity for T-cells in R. Absolute numbers of NK cells increased significantly in NR at EOC3 and EOC6, while this was significantly reduced in R at EOC3 and EOC6 compared to post-ASCT (p<0.01). This indicates that immunogenic cell death of NK cells appear to occur in the R in response to treatment. Total RNA-sequencing studies indicated that minimal changes were induced between pre-ASCT and EOC3 in NR (6 genes), while comparison of R (Pre-ASCT) to EOC3 and EOC6 demonstrated that 2482 and 2814 genes, respectively, were regulated. A total of 984 genes were differentially regulated in both EOC3 and EOC6 compared to pre-ASCT. Notably, HMGB1, a critical mediator of immunogenic cell death was significantly upregulated in R at EOC3 compared to pre-ASCT sample, consistent with our observations of NK cell proportions. Further analysis revealed that a total of 13 genes were differentially regulated in R at Post-ASCT, EOC3 and EOC6 compared to the pre-ASCT. In conclusion, we present potential pharmacodynamic biomarkers of clinical benefit (acetylation of histones, CD4/CD8 ratio and changes in immune subsets) using PBMC. A subset of genes that can be assessed from whole BM biopsy are also being assessed to determine a genetic signature for response. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Proteomic Characterization of the Dynamics of Ischemic Stroke in Mice.

Novel therapies and biomarkers are needed for the treatment of acute ischemic stroke (AIS). This study aimed to provide comprehensive insights into the dynamic proteome changes and underlying molecular mechanisms post-ischemic stroke. TMT-coupled proteomic analysis was conducted on mouse brain cortex tissue from five time points up to 4 weeks poststroke in the distal hypoxic-middle cerebral artery occlusion (DH-MCAO) model. We found that nearly half of the detected proteome was altered following stroke, but only ∼8.6% of the changes were at relatively large scales. Clustering on the changed proteome defined four distinct expression patterns characterized by temporal and quantitative changes in innate and adaptive immune response pathways and cytoskeletal and neuronal remodeling. Further analysis on a subset of 309 “top hits”, which temporally responded to stroke with relatively large and sustained changes, revealed that they were mostly secreted proteins, highly correlated to different cortical cytokines, and thereby potential pharmacodynamic biomarker candidates for inflammation-targeting therapies. Closer examination of the top enriched neurophysiologic pathways identified 57 proteins potentially associated with poststroke recovery. Altogether, our study generated a rich dataset with candidate proteins worthy of further validation as biomarkers and/or therapeutic targets for stroke. The proteomics data are available in the PRIDE Archive with identifier PXD025077.

A novel serological pharmacodynamic biomarker assessing type II collagen degradation in osteoarthritis patients

Purpose: A major hallmark of osteoarthritis (OA) is the altered cartilage metabolism and turnover. During cartilage erosion, type II collagen is cleaved by matrix metalloproteinases (MMPs) generating new protein fragments, called neo-epitopes. These fragments are released into the circulation and may reflect disease activity, and potentially be utilized for drug development as pharmacodynamic markers. Currently, there is a lack of type II collagen biomarkers reflecting pharmacologic responses to therapeutic interventions in OA. A novel immunoassay detecting a neo-epitope fragment of type II collagen (T2CM), cleaved by MMP-1 and MMP-13, has recently been developed. The assay is shown to be technically robust and highly specific for the T2CM neo-epitope. Studies have indicated that MMP-13 plays a critical role in type II collagen degradation in articular cartilage in OA and hence, T2CM may serve as a potential pharmacodynamic biomarker candidate in OA. The aim of this study was to evaluate the pharmacodynamic property of T2CM in OA patients treated with oral salmon calcitonin compared to placebo. Methods: T2CM levels were assessed in serum samples in a subset of the SMC study, a randomized, double-blind, multi-center, placebo-controlled study where knee OA patients (n=50) received 0.8 mg oral salmon calcitonin treatment twice daily for 24 months and compared to placebo (n=57). Serum samples were collected at baseline and after 1, 6, 12, and 24 months of treatment. Study samples were collected after informed consent and approved by local ethical committee in compliance with the Helsinki declaration of 1997. The statistical analysis included two-way ANOVA with Sidak’s post hoc test for difference between multiple comparisons of T2CM levels between the OA patients receiving salmon calcitonin treatment and placebo. Data were adjusted for differences in body mass index (BMI), sex, and age. Results: The mean age of OA patients treated with oral salmon calcitonin was 63.9 years (±SD 6.9) and 34% were female, and mean age of the placebo group was 63.1 years (±SD 6.1) and 45% female. The mean BMI of the treatment group and placebo was 30.2 (SD±5.1) and 27.9 (SD±3.3), respectively. T2CM levels were significantly decreased in OA patients receiving salmon calcitonin compared to the placebo group after 1, 6, and 24 months of treatment (p=0.0285; mean -61.49 ±SD 9.10 vs -33.19 ±SD 8.50, p=0.084; mean -66.35 ±SD 8.90 vs -41.46 ±SD 8.31, p=0.0035; mean -61.73 ±SD 9.49 vs -22.19 ±SD 8.76) (Fig.1). Conclusions: The novel T2CM assay measured significantly decreased levels of T2CM in OA patients treated with salmon calcitonin compared to placebo. This suggests that T2CM may have potential as a pharmacodynamic biomarker for type II collagen degradation. Currently, no disease-modifying drugs exist in OA. A validated pharmacodynamic biomarker for type II collagen degradation could promote the development of new disease-modifying OA drugs targeting cartilage repair or degradation.

Abstract PR-04: Effect of breast cancer chemoprevention on a convolutional neural network-based mammographic evaluation using a mammographic dataset of women with atypical hyperplasia, lobular or ductal carcinoma in situ

Abstract Background: Chemoprevention with anti-estrogens such as tamoxifen has been shown to lower mammographic density (MD), a strong predictor of breast cancer risk. However, measurement of MD is limited by variability in radiologists’ interpretations. We developed a novel, fully-automated convolutional neural network (CNN)-derived mammographic evaluation that is a more accurate predictor of breast cancer risk than MD. We evaluated whether chemoprevention with anti-estrogens is associated with a significant change in CNN breast cancer risk among women with atypical hyperplasia (AH), lobular or ductal carcinoma in situ (LCIS/DCIS). Methods: We conducted a retrospective cohort study using serial mammograms from women diagnosed with AH, LCIS, or DCIS at Columbia University Irving Medical Center (CUIMC) between 2007 and 2015. We collected mammograms at baseline (at diagnosis of AH/LCIS/DCIS or prior to initiation of chemoprevention) and 3-5 years follow-up. We extracted information from the electronic health record on age, race/ethnicity, menopausal status, body mass index (BMI), and chemoprevention uptake (yes/no). Briefly, each mammogram was normalized as a map of z-scores and resized to an input image size of 256 × 256. Then a contracting and expanding fully convolutional CNN architecture was composed entirely of 3 × 3 convolutions, a total of four strided convolutions instead of pooling layers, and symmetric residual connections. L2 regularization and augmentation methods were implemented to prevent over-fitting. CNN risk score was expressed as a continuous variable (0-1). We used 2-sample t-test to compare change in CNN risk score from baseline to follow-up among women who took chemoprevention compared to those who did not. We conducted multivariable linear regression adjusting for known breast cancer risk factors (age, BMI, menopausal status, race/ethnicity) to determine whether receipt of chemoprevention was associated with change in CNN risk score. Results: Among 728 evaluable women, mean age was 60.4 years (SD, 11.1), 70.4% were postmenopausal, and 248 (34.1%) received chemoprevention with anti-estrogens while 480 (65.9%) did not. Women who received chemoprevention compared to those who did not had a greater mean change in CNN risk score from baseline to 3-5 years of follow-up, -0.069 (SD, 0.278) and -0.019 (SD, 0.244), respectively (p=0.014). In multivariate analysis, women who received chemoprevention compared to those who did not had a 0.038 point greater decrease in CNN risk score (p=0.085, 95% confidence interval [CI]= -0.081, +0.005). Conclusions: We demonstrated that our CNN-based mammographic evaluation is modifiable with anti-estrogen therapy among high-risk women. Future studies should determine whether changes in CNN risk score are associated with the development of breast cancer, in order to further evaluate the CNN mammographic evaluation as a potential pharmacodynamic biomarker of response to breast cancer chemoprevention. Citation Format: Julia E. McGuinness, Vicky Ro, Aishwarya Anuraj, Haley Manley, Simukayi Mutasa, Richard Ha, Katherine D. Crew. Effect of breast cancer chemoprevention on a convolutional neural network-based mammographic evaluation using a mammographic dataset of women with atypical hyperplasia, lobular or ductal carcinoma in situ [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PR-04.

Trials in Progress: A Phase I Study to Evaluate the Safety and Pharmacokinetic Profiles of CB-5339 in Participants with Relapsed/Refractory Acute Myeloid Leukemia or Relapsed/Refractory Intermediate or High-Risk Myelodysplastic Syndrome

Trials in Progress: A Phase I Study to Evaluate the Safety and Pharmacokinetic Profiles of CB-5339 in Participants with Relapsed/Refractory Acute Myeloid Leukemia or Relapsed/Refractory Intermediate or High-Risk Myelodysplastic Syndrome