Potential Pharmacodynamic Biomarkers Research Articles

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

Abstract 4808: ONC201 decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, intregrated stress response and tumor cell death

Abstract Brain tumors are one of the most lethal cancers in children and young adults. We previously reported that first-in-class small-molecule imipridone ONC201 can antagonize dopamine receptor D2 (DRD2), inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce growth arrest or cell death in tumor cells. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant glioma patients. We hypothesized that ONC201 may synergize with radiotherapy and temozolomide in brain tumors. Glioblastoma (GBM: SNB19, T98G and U251), diffuse intrinsic pontine glioma (DIPG: SU-DIPG-IV and SU-DIPG-36) and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines were tested using cell viability or colony formation assays with ONC201 up to 20 μM alone or in combination with radiotherapy up to 8 Gy and temozolomide up to 100 μM. We observed synergy between ONC201 and radiation and temozolomide by multiple assays with the best combination index of 0.31 under the treatment of 0.625 μM ONC201, 25 μM TMZ and 4 Gy irradiation in cell viability assay. We observed induction of PKA substrate phosphorylation as a marker of DRD2 antagonism, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death in treated brain tumor cells. Mitochondrial dysfunction induced by ONC201 was greatly reflected in reduction of maximal cell respiration in brain tumor cell lines. ClpXP is a proteolytic machine in mitochondria comprising of the protein chaperone ClpX and protease ClpP, a recently described binding target of ONC201. We have observed that ONC201 induced cytotoxicity depends on ClpP and knockdown of ClpP strongly protects multiple human cancer cell lines from ONC201-mediated (but not other drugs, e.g. temozolomide) cytotoxicity. The ClpX protein level decreased dramatically upon ONC201 treatment. We are exploring the potential interplay between ClpP, dopamine receptors, the ISR and TRAIL signaling pathways after single agent or combinatorial treatments. Our data suggests that ONC201 may be combined synergistically with temozolomide or radiation to address gliomas, along with potential pharmacodynamic biomarkers. The results implicate ClpX and ClpP in downstream cell death induction following ONC201 treatment of brain tumor cells. Citation Format: Lanlan Zhou, Howard P. Safran, Michael J. Glantz, Wafik S. El-Deiry. ONC201 decreases protein chaperone ClpX to unleash mitochondrial protease ClpP activity, intregrated stress response and tumor cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4808.

CYP3A5 Genotype as a Potential Pharmacodynamic Biomarker for Tacrolimus Therapy in Ulcerative Colitis in Japanese Patients.

Tacrolimus has been used to induce remission in patients with steroid-refractory ulcerative colitis. It poses a problem of large individual differences in dosage necessary to attain target blood concentration and, often, this leads to drug inefficacy. We examined the difference in mRNA expression levels of ATP binding cassette transporter B1 (ABCB1) between inflamed and non-inflamed tissues, and the influence of CYP3A5 genotype on tacrolimus therapy. The mRNA expression of CYP3A4 in colonic mucosa and that of cytochrome p450 3A5 (CYP3A5) and ABCB1 in inflamed and non-inflamed areas were examined in 14 subjects. The mRNA expression levels of CYP3A5 were higher than that of CYP3A4. The mRNA expression of ABCB1 was lower in the inflamed than in the non-inflamed mucosa, despite that of CYP3A5 mRNA level being not significantly changed. Hence, the deterioration of the disease is related to the reduction of the barrier in the inflamed mucosa. The relationship between CYP3A5 genotype and blood concentration, dose, and concentration/dose (C/D) ratio of tacrolimus in 15 subjects was studied. The tacrolimus dose to maintain equivalent blood concentrations was lower in CYP3A5*3/*3 than in CYP3A5*1 carriers, and the C/D ratio was significantly higher in the latter. Thus, CYP3A5 polymorphism information played a role in determining the initial dose of tacrolimus. Furthermore, since the effect of tacrolimus appears earlier in CYP3A5*3/*3 than in CYP3A5*1/*1 and *1/*3, it seems necessary to change the evaluation time of therapeutic effect by CYP3A5 genotype. Additionally, the relationship between CYP3A5 genotype and C/D ratio of tacrolimus in colonic mucosa was investigated in 10 subjects. Tacrolimus concentration in the mucosa was two-fold higher in CYP3A5*3/*3 than in CYP3A5*1 carriers, although no significant difference in tacrolimus-blood levels was observed. Therefore, the local concentration of tacrolimus affected by CYP3A5 polymorphism might be related to its therapeutic effect.

Phase I study of AMG 160, a half-life extended bispecific T-cell engager (HLE BiTE immune therapy) targeting prostate-specific membrane antigen, in patients with metastatic castration-resistant prostate cancer (mCRPC).

TPS5590 Background: Prostate-specific membrane antigen (PSMA) is a clinically validated therapeutic target for the imaging and treatment of mCRPC. AMG 160 is an HLE BiTE immune therapy designed to redirect T cells to cancer cells by binding to PSMA on cancer cells and CD3 on T cells. BiTE immune therapy leads to direct tumor cell killing, T-cell activation and expansion, and the creation of a pro-inflammatory tumor microenvironment. Combining AMG 160 with a PD-1 inhibitor may enhance antitumor activity by enabling sustained T-cell-dependent killing of tumor cells in the inflamed tumor microenvironment. Methods: NCT03792841 is a phase I study of AMG 160 as monotherapy (part 1) and in combination with pembrolizumab (part 2) in men with histologically/cytologically confirmed mCRPC who are refractory to a novel hormonal therapy (abiraterone, enzalutamide, and/or apalutamide) and have failed 1–2 taxane regimens (or are medically unsuitable or have refused taxanes), who have ongoing castration with total serum testosterone ≤ 50 ng/dL, and have evidence of progressive disease. Patients who received prior PSMA radionuclide therapy may be eligible. Patients with CNS metastases, leptomeningeal disease, spinal cord compression, or active autoimmune disease will be excluded. Primary objectives are to evaluate safety and tolerability and determine the maximum tolerated dose (MTD) or recommended phase II dose (RP2D) of AMG 160 given as monotherapy or in combination with pembrolizumab. Secondary objectives are to characterize pharmacokinetics and preliminary antitumor activity. Exploratory objectives include evaluation of potential pharmacodynamic and patient selection biomarkers, immunogenicity, and patient-reported pain and functional outcomes. The part 1 dose exploration will determine the MTD/RP2D of AMG 160. The part 1 dose expansion will confirm the safety and tolerability of the MTD/RP2D. The part 2 dose exploration will estimate the MTD/RP2D of AMG 160 in combination with pembrolizumab. Evaluation of preliminary antitumor activity will be based on RECIST 1.1 with Prostate Cancer Working Group 3 modifications, PSA response, CTC response, progression-free survival (radiographic and PSA), and overall survival. PSMA PET/CT and FDG PET/CT imaging will be used for evaluation of exploratory objectives. The study opened in February 2019 and is currently recruiting patients into both part 1 and part 2. Clinical trial information: NCT03792841 .

IGM-8444 as a potent agonistic Death Receptor 5 (DR5) IgM antibody: Induction of tumor cytotoxicity, combination with chemotherapy and in vitro safety profile.

3595 Background: Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that multimerizes when bound to its ligand, TNF-related apoptosis inducing ligand (TRAIL), to activate the extrinsic apoptotic pathway. DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have generally been unsuccessful due to toxicity or lack of efficacy. We have developed a multivalent IgM DR5 agonist, IGM-8444, that multimerizes DR5 to selectively and potently induce tumor cell apoptosis while maintaining tolerability. Methods: IGM-8444 is an engineered, pentameric IgM antibody with 10 binding sites specific for DR5. Human tumor cell lines or hepatocytes were evaluated in vitro for dose dependent IGM-8444 induced cytotoxicity. The efficacy of IGM-8444 was evaluated with or without chemotherapy, in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse tumor models, with IGM-8444 administered at various dose levels and schedules when tumors reached approximately 100 mm3. Sera and tumors were analyzed for biomarkers of tumor apoptosis. Results: In vitro cytotoxicity assays identified IGM-8444 activity across cell lines from 18 solid and hematologic malignancies. In IGM-8444 partially resistant cell lines, combination with chemotherapy or a Bcl2 inhibitor enhanced in vitro cytotoxicity. IGM-8444 was efficacious as a monotherapy in CDX and PDX tumor models including colorectal, lung, and gastric indications. In a gastric PDX model, IGM-8444 induced complete and durable dose-dependent tumor regressions. In vivo, combination of IGM-8444 with standard-of-care chemotherapies, such as irinotecan, led to enhanced efficacy. IGM-8444 administration increased markers of tumor apoptosis, identifying potential clinical pharmacodynamic biomarkers. At doses several log-fold higher than efficacious doses, IGM-8444 demonstrated a favorable single agent in vitro safety profile, with little to no in vitro cytotoxicity observed using primary human hepatocytes from multiple donors. Conclusions: These data support the clinical development of IGM-8444 in both solid and hematological malignancies as a single agent and in combination with standard of care therapy. IGM-8444 is projected for IND filing in 2020.

Validation of serum neurofilaments as prognostic and potential pharmacodynamic biomarkers for ALS.

To identify preferred neurofilament assays and clinically validate serum neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) as prognostic and potential pharmacodynamic biomarkers relevant to amyotrophic lateral sclerosis (ALS) therapy development. In this prospective, multicenter, longitudinal observational study of patients with ALS (n = 229), primary lateral sclerosis (n = 20), and progressive muscular atrophy (n = 11), biological specimens were collected, processed, and stored according to strict standard operating procedures (SOPs). Neurofilament assays were performed in a blinded manner by independent contract research organizations. For serum NfL and pNfH measured using the Simoa assay, there were no missing data (i.e., technical replicates below the lower limit of detection were not encountered). For the Iron Horse and Euroimmun pNfH assays, such missingness was encountered in ∼4% and ∼10% of serum samples, respectively. Mean coefficients of variation for NfL in serum and CSF were both ∼3%. Mean coefficients of variation for pNfH in serum and CSF were ∼4%-5% and ∼2%-3%, respectively, in all assays. Baseline serum NfL concentration, but not pNfH, predicted the future Revised ALS Functional Rating Scale (ALSFRS-R) slope and survival. Incorporation of baseline serum NfL into mixed effects models of ALSFRS-R slopes yields an estimated sample size saving of ∼8%. Depending on the method used to estimate effect size, use of serum NfL (and perhaps pNfH) as pharmacodynamic biomarkers, instead of the ALSFRS-R slope, yields significantly larger sample size savings. Serum NfL may be considered a clinically validated prognostic biomarker for ALS. Serum NfL (and perhaps pNfH), quantified using the Simoa assay, has potential utility as a pharmacodynamic biomarker of treatment effect.

UHPLC Q-Exactive MS-based spleen metabolomics and lipidomics to explore the effect mechanisms of Danggui Buxue Decoction in anemia mice

Danggui Buxue Decoction (DBD), a famous traditional Chinese medicine (TCM), is often used to treat anemia in China. However, its underlying therapeutic mechanism is unclear. Through the analysis of body weight, spleen and thymus indexes, peripheral blood routine and pathological section of femur, it was obviously that DBD could significantly improve acetylphenylhydrazine (APH) + cyclophosphamide (CTX) induced anemia mice in the present work. Ultra high performance liquid chromatography coupled with quadrupole – Exactive mass spectrometry (UHPLC Q-Exactive MS) based metabolomics and lipidomics was further utilized to screen out differential spleen metabolites associated with DBD treatment. A total of 26 differential metabolites including 8 polar metabolites and 18 lipids were firstly obtained to relate with anemia mice. 7 polar metabolites and 10 lipids among them were reversed by DBD, which the regulation of pyrimidine metabolism and glycerophospholipid metabolism were mainly associated to the anti-anemia effect of DBD based on MetaboAnalyst analysis. Through random forest analysis (RF), ROC analysis and pearson matrix correlation, three metabolites, cytosine, uracil and PC (o-16:1(9Z)/20:0), were further screened out as the potential pharmacodynamic biomarkers associated with the efficacy of DBD. This study provided a methodological reference for the study of the mechanism of TCM.

The novel proautophagy anticancer drug ABTL0812 potentiates chemotherapy in adenocarcinoma and squamous nonsmall cell lung cancer.

Around 40% of newly diagnosed lung cancer patients are Stage IV, where the improvement of survival and reduction of disease-related adverse events is the main goal for oncologists. In this scenario, we present preclinical evidence supporting the use of ABTL0812 in combination with chemotherapy for treating advanced and metastatic Nonsmall cell lung adenocarcinomas (NSCLC) and squamous carcinomas. ABTL0812 is a new chemical entity, currently in Phase 1b/2a clinical trial for advanced squamous NSCLC in combination with paclitaxel and carboplatin (P/C), after successfully completing the first-in-human trial where it showed an excellent safety profile and signs of efficacy. We show here that ABTL0812 inhibits Akt/mTOR axis by inducing the overexpression of TRIB3 and activating autophagy in lung squamous carcinoma cell lines. Furthermore, treatment with ABTL0812 also induces AMPK activation and ROS accumulation. Moreover, combination of ABTL0812 with chemotherapy markedly increases the therapeutic effect of chemotherapy without increasing toxicity. We further show that combination of ABTL0812 and chemotherapy induces nonapoptotic cell death mediated by TRIB3 activation and autophagy induction. We also present preliminary clinical data indicating that TRIB3 could serve as a potential novel pharmacodynamic biomarker to monitor ABTL0812 activity administered alone or in combination with chemotherapy in squamous NSCLC patients. The safety profile of ABTL0812 and its good synergy with chemotherapy potentiate the therapeutic potential of current lines of treatment based on chemotherapy regimens, arising as a promising option for improving these patients therapeutic expectancy.

Nuclear factor of activated T cells as potential pharmacodynamic biomarker for the risk of acute and subclinical rejection in de novo liver recipients.

Nuclear factor of activated T cell-regulated gene expression (NFAT-RGE) has been proposed as a pharmacodynamic biomarker for tacrolimus (Tac) and cyclosporine (CsA). Our aim was to evaluate the role of NFAT-RGE in modulating intralymphocytary IL-2 and IFN-γ expression and its clinical utility as an early non-invasive predictive biomarker for the risk of acute rejection (AR) and infection in de novo liver transplant (LT) recipients. Fifty-six LT recipients treated with Tac or CsA [with and without mycophenolate mofetil (MMF)] were included: 30 free of rejection or infection, 11 rejectors (T cell-mediated acute rejection), 5 with subclinical rejection (SCR) and 10 with cytomegalovirus (CMV) infection. Within the first 3months after transplantation, NFAT-RGE of IL-2, IFN-γ and GM-CSF and intralymphocytary synthesis of IL-2 and IFN-γ were evaluated by real-time PCR and flow cytometry respectively. A significant increase in NFAT-RGE was observed in patients who experienced TCMAR (75% [42-100%]) or SCR (41% [18-78%]) compared with patients without rejection or infection (14% [2-23%]). Positive correlations between the %NFAT-RGE-IFN and both the %CD8CD69IFN-γ and %CD4CD69IFN-γ and between the %NFAT-RGE-IL2 and the %CD8CD69IL2 were observed. NFAT-RGE was significantly lower in CMV+ patients than in non-infected patients. Finally, an inverse correlation between the Tac or CsA concentration and inhibition of NFAT-RGE were observed. Sequential post-transplantation NFAT-RGE monitoring combined with intralymphocytary IL-2 and IFN-γ before transplantation and at the first and third month post-transplantation may be key predictive and diagnostic biomarkers for the risk of TCMAR and SCR and better guide CNi therapy in LT patients.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Abstract B108: 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors

Abstract Aberrantly activated PI3K/mTOR signaling is frequently implicated as an oncogenic driver in human cancer. In response to nutrient and growth factor stimuli, mTOR complex 1 (mTORC1) regulates cell growth and cap-dependent protein translation through phosphorylation and activation of S6K, and through phosphorylation and inactivation of the translational repressor 4EBP1. While suppression of mTORC1 activity causes rapid dephosphorylation of its substrates S6K and 4EBP1, it has also been reported that the translational repressor 4EBP3 is transcriptionally induced during prolonged mTORC1 inhibition.1 We have designed a series of complete and selective bi-steric inhibitors of mTORC1 by covalently linking rapamycin, a selective yet incomplete mTORC1 inhibitor, with mTOR kinase active-site inhibitors. These compounds potently and durably suppress phosphorylation of S6K and 4EBP1, induce growth suppression and apoptosis in multiple cancer cell lines, and cause tumor growth inhibition in xenograft models. In the present study we evaluated the utility of 4EBP3 mRNA expression as a pharmacodynamic (PD) biomarker of mTORC1 inhibition by our bi-steric inhibitors. We further interrogated whether tumor mTORC1 inhibition and anti-tumor efficacy could be predicted by monitoring this PD marker in surrogate tissue. First, we demonstrated that 4EBP3 mRNA and protein levels increased in response to mTORC1 inhibition in a concentration- and time-dependent manner in MDA-MB-468, MCF-7, and HCC1954 breast carcinoma cells. Kinetic studies in which we compared the response of bi-steric inhibitors to that of short duration-of-action active-site inhibitors revealed that 4EBP3 induction requires sustained mTORC1 inhibition, as loss of mTORC1 inhibition correlated with a rapid return to baseline 4EBP3 expression. Treatment with inhibitors of varying potency against mTORC1 also demonstrated a strong correlation between magnitude of cell growth inhibition and 4EBP3 mRNA induction. Additionally, suppression of tumor 4EBP1 phosphorylation and induction of 4EBP3 mRNA was observed in MCF-7 and HCC9154 tumor xenografts following in vivo administration of bi-steric inhibitors with a concomitant induction of murine 4ebp3 in peripheral blood mononuclear cells (PBMCs). Initial studies indicate that ex vivo exposure of human PBMCs to a mTORC1 inhibitor also promotes 4EBP3 mRNA expression. Together, these data suggest that 4EBP3 could provide a sensitive mRNA-based in vivo biomarker of prolonged mTORC1 inhibition, as is the case following administration of a single dose of a bi-steric mTORC1 inhibitor. The concordance between the responses in tumor and PBMC suggests it may be possible to monitor tumor mTORC1 inhibition by monitoring 4EBP3 in surrogate tissue. In summary, 4EBP3 mRNA represents a potential PD biomarker of mTORC1 inhibitor response as an alternative to measurement of the phosphorylation status of mTORC1 substrates. 1. Tsukumo, T., et al. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3. Nat Commun 7:11776. doi: 10.1038/ncomms11776 (2016). Citation Format: Bianca J. Lee, Nuntana Dinglasan, Tram Nguyen, Ed Lorenzana, Stacy Wilson, G. Leslie Burnett, James B. Aggen, Robert J. Nichols, Mallika Singh, David Wildes, Jaqueline A.M. Smith. 4EBP3 mRNA as a biomarker of therapeutic response to treatment with mTORC1 inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B108. doi:10.1158/1535-7163.TARG-19-B108

Identification of a Pharmacodynamic Biomarker for SEL24/MEN1703, a First-in-Class Dual PIM/FLT3 Kinase Inhibitor for Acute Myeloid Leukemia, and Its Implementation in the First-in-Human Study CLI24-001

SEL24/MEN1703 is a first-in-class, orally available, dual PIM/FLT3 kinase inhibitor currently investigated in patients with Acute Myeloid Leukemia (AML) in the first-in-human study CLI24-001 (NCT03008187). PIM and FLT3 kinases are considered to play an important role in AML and are inhibited by SEL24/MEN1703. Moreover, there is evidence that inhibition of PIM kinases might contribute to overcoming acquired resistance induced by approved FLT3 inhibitors1. In AML, different signal transducers in the FLT3 pathway are substrates of kinases. Therefore, their phosphorylation levels might be modulated by kinase inhibitors and may be exploited as a potential pharmacodynamic biomarker in clinical development. In particular, phosphorylation of S6, 4E-BP1, and STAT5 is regulated by both FLT3 and PIM1/2. Thus, the objective of this investigation was to identify the most promising pharmacodynamic biomarker/s for implementation in the clinical trials of SEL24/MEN1703. Initially, we assessed the in vitro cytotoxic effect of SEL24/MEN1703 in a panel of 26 AML cell lines harboring different genetic mutations, to identify suitable cell lines for subsequent experiments. In the selected panel of AML cell lines, SEL24/MEN1703 resulted in the inhibition of phosphorylation of S6, 4-EBP1 and STAT5 as measured by immunoblotting. Notably, the reduction in phosphorylated S6 (pS6) in response to SEL24/MEN1703 was particularly evident. Since SEL24/MEN1703 displays a broad cytotoxic activity in AML cell lines, we clustered sensitive and resistant cell lines considering 0.5 μM as the IC50 cut-off value. Then, we investigated the relationship between SEL24/MEN1703 cytotoxic activity in AML cell lines and the inhibition of the above mentioned phosphorylated proteins in a 24-hour cytotoxic assay, showing a correlation between IC50 and the reduction of pS6 (Pearson correlation coefficient: -0.6905, R2= 0.477). To further confirm the in vitro data, SEL24/MEN1703 ability to modulate phosphoproteins was assessed also in xenograft mice bearing MOLM-16 cell line. The phosphorylation status of S6, 4E-BP1 and STAT5 was analyzed by immunoblot in tumor tissues from mice treated at 25 mg/kg of SEL24/MEN1703 at baseline and at 4, 8, and 16 hours after treatment. Results showed that also in vivo, SEL24/MEN1703 administration resulted in a decrease of pS6, with maximum reduction in this parameter observed 4 hours after the administration of the investigational compound. Based on these results, pS6 was identified as the pharmacodynamic biomarker to be implemented in the CLI24-001 clinical trial. Among different available methods, flow cytometry was selected as the preferred platform to analyze patient samples, because of its ability to provide quantitative assessment of cellular events and pharmacodynamic evaluation in a selected, relevant cell subpopulation, such as the AML blast cells. The assessment of pS6 in the clinical trial is planned both at baseline and at cycle 1 day 14 for whole blood and bone marrow. In addition, pS6 levels will be measured in whole blood at additional time points during treatment cycles. We have implemented the measurement of pS6 in the CLI24-001 trial, and pS6 levels as well as their relationship with the main pharmacokinetic parameters in patients treated with SEL24/MEN703 at 100 and 125 mg will be presented. 1Green A.S. et al., Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia, Sci Adv, 2015 Disclosures Tomirotti: Menarini Ricerche S.p.A.: Employment. Merlino:Menarini Ricerche S.p.A.: Employment. Fiascarelli:Menarini Ricerche S.p.A.: Employment. Baldini:Menarini Ricerche S.p.A.: Employment. Tagliavini:Menarini Ricerche S.p.A: Employment. Borella:Menarini Ricerche S.p.A.: Employment. Mazan:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Bressan:Menarini Ricerche S.p.A.: Employment. Pellacani:Menarini Ricerche S.p.A.: Employment; Amgen: Equity Ownership. Salerno:Menarini Ricerche S.p.A.: Employment. Binaschi:Menarini Ricerche S.p.A.: Employment. Bellarosa:Menarini Ricerche S.p.A.: Employment.

A Phase I/II Study of NMS-03592088, a FLT3, KIT and CSF1R Inhibitor, in Patients with Relapsed or Refractory AML or CMML

A Phase I/II Study of NMS-03592088, a FLT3, KIT and CSF1R Inhibitor, in Patients with Relapsed or Refractory AML or CMML

EXTH-57. PRECLINICAL COMBINATION OF ONC201 WITH RADIOTHERAPY OR TEMOZOLOMIDE IN GBM, DIPG AND ATRT CELL LINES RESULTS IN DOPAMINE RECEPTOR ANTAGONISM, ATF4 INDUCTION AND CELL DEATH

Abstract CNS tumors are one of the most lethal cancers in children and young adults. We previously reported that first-in-class small-molecule imipridone ONC201 can antagonize dopamine receptor D2 (DRD2), inactivate ERK/AKT, induce the integrated stress response (ISR), upregulate pro-apoptotic TRAIL receptor DR5, deplete cancer stem cells, and induce growth arrest or cell death in tumor cells. ONC201 crosses the blood-brain barrier and has induced durable tumor regressions in adult and pediatric H3K27M-mutant glioma patients. We hypothesized that ONC201 may synergize with radiotherapy or temozolomide in brain tumors. Glioblastoma (GBM: SNB19, T98G and U251), diffuse intrinsic pontine glioma (DIPG: SF8628) and atypical teratoid rhabdoid tumor (ATRT: BT-12, BT-16) cell lines were tested using cell viability or colony formation assays with ONC201 up to 20 μM alone or in combination with radiotherapy up to 8 Gy or temozolomide up to 100 μM. We observed synergy between ONC201 and radiation or temozolomide by multiple assays. We observed induction of PKA substrate phosphorylation as a marker of DRD2 antagonism, induction of ATF4 as a marker of ISR activation, and multiple markers of cell death in treated brain tumor cells. We are evaluating the relevance of mitochondrial protease ClpP, a recently described binding target of ONC201, in ONC201-induced glioma cell death. We have observed that knockdown of ClpP strongly protects multiple human cancer cell lines from ONC201-mediated cytotoxicity. Thus, we are exploring the potential interplay between ClpP, dopamine receptors, the ISR and TRAIL signaling pathways after single agent or combinatorial treatments. Our data suggests that ONC201 may be combined synergistically with temozolomide or radiation to address gliomas, along with potential pharmacodynamic biomarkers.

1215P - T cell repertoire sequencing reveals dynamics of response to dendritic cell vaccine plus dasatinib for checkpoint blockade resistant metastatic melanoma

BackgroundWe recently conducted an open- label, single-center, prospective randomized Phase II clinical trial for HLA-A2+ patients with advanced-stage melanoma (NCT01876212) at the University of Pittsburgh Hillman Cancer Center. Patients received ID injections of Type-1-polarized, autologous DC loaded with a mixture of peptides derived from six tumor-associated vascular antigens (DLK1, EPHA2, HBB, NRP1, RGS5, TEM1) combined +/- daily oral administration of dasatinib (70mg BID) as an immune adjuvant/conditioning agent. Here we report an exploratory analysis of T cell repertoire profiling of longitudinally-sampled peripheral blood leukocytes (PBL) to define potential pharmacodynamic and response biomarkers. MethodsTotal RNA was extracted from pre- and post-treatment PBL from 13 therapy recipients (6 responders, 7 non-Responders) including extended longitudinal samples for four responders. TCRB sequencing was performed via the Oncomine TCRB-LR assay using 25ng total RNA as input. We evaluated T cell clonal expansion and TCR convergence as potential biomarkers of response. ResultsTCR convergence values were elevated in pretreatment PBL of responders compared to non-responders (mean frequency .012 vs .006; p=.01, Wilcoxon), and remained elevated in responders up to 25 weeks post treatment. TCR evenness (normalized Shannon entropy) decreased at week 5 compared to baseline (p=.01, one-sided student’s t-test), indicating increased clonal expansion following treatment. ConclusionsThese data suggest that peripheral blood TCRB convergence may serve as a predictive or prognostic biomarker for response to dendritic cell-based immunotherapy. Our finding of increased T cell clonal expansion at week 5 of treatment supports the notion that TCR sequencing may serve as a tool for the measurement of pharmacodynamic markers of therapeutic agent activity. Ongoing and future studies will further clarify the utility of TCR convergence and clonal expansion as immune repertoire biomarkers. Clinical trial identificationNCT01876212. Legal entity responsible for the studyUniversity of Pittsburgh School of Medicine & Hillman Cancer Center. FundingThermo Fisher Scientific. DisclosureL. Quagliata: Full / Part-time employment: Thermo Fisher Scientific. T. Looney: Full / Part-time employment: Thermo Fisher Scientific. D. Topacio-Hall: Full / Part-time employment: Thermo Fisher Scientific. G. Lowman: Full / Part-time employment: Thermo Fisher Scientific. All other authors have declared no conflicts of interest.

817 Identification of Biomarkers and Mechanistic Insight for Upadacitinib in Crohn’s Disease: Serum Inflammatory Mediator Analysis From the Phase 2b CELEST Study

INTRODUCTION: We report pharmacodynamic profiling of serum inflammatory mediators in CELEST (patients) pts with the aim of understanding UPA mechanism of action in CD and linking biomarkers to intestinal inflammation, abdominal pain (AP), and stool frequency (SF). METHODS: The CELEST Ph2 study evaluated the efficacy/ safety of multiple dosing regimens of UPA vs PBO in adults with moderately to severely active CD and inadequate response or intolerance to immunomodulators and/or TNF inhibitors.1,2 Serum samples from baseline (BL) visit and end of induction period (Wk16) were analyzed by OLINK® inflammation panel (92 proteins) and Singulex immunoassay for interleukin-17A(IL-17A), IL-17F, IL-22, and IL-23p19. Protein level changes (BL–Wk16) were analyzed by an ANCOVA model where BL protein level was adjusted as a covariate and treatment effect was considered significant when a false discovery rate < 0.10 was observed after multiplicity adjustment on F-test P-values. Spearman rank correlation coefficients(rs) determined relationships between BL levels or BL–Wk16 level changes of serum biomarkers (also including serum C-reactive protein and fecal calprotectin) and improvements in Simple Endoscopic Score for CD (SES-CD), AP, and SF. RESULTS: Paired BL and Wk16 serum samples were available from 104 pts (UPA 3 mg twice daily [BID], n = 18; 6 mg BID, n = 19; 12 mg BID, n = 13; 24 mg BID, n = 18; 24 mg once daily, n = 18; and PBO, n = 18). UPA treatment significantly reduced expression of serum pro-inflammatory mediators (Table). that are associated with immune cell migration, type I/II interferon (IFN) responses, T cell activation, T helper cell-1 (Th1) responses, and CD8+ T cell responses. UPA treatment did not significantly modulate expression of IL-23/Th17-related serum cytokines in CD. Correlations were observed for BL–Wk16 changes in serum proteins and SES-CD, AP, or SF. Importantly, oncostatin-M reductions correlated with SES-CD (rs = 0.35, P = 0.02) and SF improvement (rs = 0.33, P = 0.04), suggesting similar drivers of these aspects of disease. AP improvement correlated with reductions in CSF-1 (rs = 0.37, P = 0.01), TNFRSF9 (rs = 0.34, P = 0.03), IL-12B (rs = 0.31, P = 0.07), CXCL9 (rs = 0.31, P = 0.07), and CD8A (rs = 0.31, P = 0.09), which are all associated with the Th1/CD8/IFN-γ pathway. CONCLUSION: This analysis of inflammatory mediators in serum of CD pts from CELEST identifies potential pharmacodynamic biomarkers and mechanistic insights for the JAK-1 inhibitor, UPA.

Pharmacometabolomic Pathway Response of Effective Anticancer Agents on Different Diets in Rats with Induced Mammary Tumors.

Metabolomics is an effective approach to characterize the metabotype which can reflect the influence of genetics, physiological status, and environmental factors such as drug intakes, diet. Diet may change the chemopreventive efficacy of given agents due to the altered physiological status of the subject. Here, metabolomics response to a chemopreventive agent targretin or tamoxifen, in rats with methylnitrosourea-induced tumors on a standard diet (4% fat, CD) or a high fat diet (21% fat, HFD) was evaluated, and found that (1) the metabolome was substantially affected by diet and/or drug treatment; (2) multiple metabolites were identified as potential pharmacodynamic biomarkers related to targretin or tamoxifen regardless of diet and time; and (3) the primary bile acid pathway was significantly affected by targretin treatment in rats on both diets, and the lysolipid pathway was significantly affected by tamoxifen treatment in rats on the high fat diet.

Abstract 2399: LAG-3/PD-L1 mAb2 can overcome PD-L1-mediated compensatory upregulation of LAG-3 induced by single-agent checkpoint blockade

Abstract FS118, currently tested in Phase I clinical trial in patients with advanced malignancies (NCT03440437), is a first-in-class bispecific antagonistic antibody (known as mAb2) targeting LAG-3 (Lymphocyte-Activation Gene 3) and PD-L1 (Programmed Death-Ligand 1), two immune checkpoint molecules that promote tumour escape from immune surveillance. Resistance to anti-PD-1 treatment is associated with upregulation of other checkpoint inhibitor receptors such as LAG-3. Dual targeting in immune checkpoint blockade (ICB) using bispecific monoclonal antibodies could potentially overcome this resistance, further increase the clinical benefit of ICB therapy and prevent relapse or resistance after immunotherapy. In a syngeneic model we used a surrogate mAb2 of FS118 (blocking both mPD-L1 and mLAG-3 binding to their receptors) to determine the effect of dual checkpoint blockade on tumour growth and to elucidate the modulation of the underlying immune mechanisms following treatment with the mAb2. Dual blockade of LAG-3 and PD-L1 with the surrogate mAb2 in the MC38 tumour-bearing mice model resulted in an increased anti-tumour activity comparable to a combination of the single agents targeting LAG-3 and PD-L1. Moreover, the mAb2 and single agent combination resulted in distinct modulations of LAG-3 and PD-L1 cell surface expression within the spleen and tumour microenvironment (TME). While both the mAb2 and combination therapy significantly reduced the number of free PD-L1 binding sites on CD4+ and CD8+ T cells in spleen and TME, total LAG-3 cell surface expression increased following treatment with the combination, whereas it reduced with mAb2 treatment. In addition, analysis of serum samples collected at various timepoints confirmed evidence of drug target engagement with increasing levels of both soluble LAG-3 (sLAG-3) and soluble PD-L1 (sPD-L1) in the mAb2 treated animals. Modulation of sPD-L1 levels in the serum of treated animals was also observed in cynomolgus monkeys treated with FS118. These data provide a strong rationale for investigating both cell surface and soluble LAG-3 and PD-L1 levels as potential pharmacodynamic biomarkers in further clinical development. Citation Format: Mustapha Faroudi, Matthew Kraman, Natalie Fosh, Claire Reader, Daniel Gliddon, Claire Seal, Christa Lucas, Alexander Koers, Mateusz Wydro, Michelle Morrow, Neil Brewis. LAG-3/PD-L1 mAb2 can overcome PD-L1-mediated compensatory upregulation of LAG-3 induced by single-agent checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2399.

Torque teno virus viremia in patients with chronic arthritis: Influence of biologic therapies

ObjectiveTorque teno virus (TTV) is a highly prevalent non-pathogenic anellovirus whose plasma levels may be a biomarker of immunosuppression. The aim of this study was to assess whether specific immune-targeting with different biologic drugs may differentially modulate TTV viremia in arthritis patients. MethodsTTV DNA load was quantified by PCR in a cross-sectional sample of 79 patients with chronic arthritis on biologic therapy (abatacept, infliximab, rituximab or tocilizumab), 31 patients treated with conventional DMARDs (methotrexate and/or leflunomide), and 54 healthy individuals. Longitudinal changes in TTV load were analysed in a second group of 59 patients at baseline and 4-months after biologic therapy. Correlations between clinical or biological characteristics of recruited patients and TTV viremia were also analysed. ResultsIn the cross-sectional study, TTV load was significantly higher in patients who received abatacept, infliximab or tocilizumab compared to healthy individuals. Patients treated with rituximab or conventional DMARDs showed TTV loads similar to healthy controls. In the longitudinal study, an increase in the TTV load was observed after anti-TNF, tocilizumab, abatacept and rituximab, but not after secukinumab therapy. Correlations between TTV load and clinical variables such as disease duration, concomitant glucocorticoid or DMARDs therapy, lymphocytes or previous infections were not found. A non-significant trend towards higher TTV load was observed in therapy responders. ConclusionPatients with chronic arthritis on biologic but not on conventional DMARD or anti-IL17 therapy have increased TTV viremia. This observation provides a basis to prospectively explore the potential value of TTV load as a potential pharmacodynamic biomarker.

The current state of biomarker research for Friedreich's ataxia: a report from the 2018 FARA biomarker meeting.

The 2018 FARA Biomarker Meeting highlighted the current state of development of biomarkers for Friedreich’s ataxia. A mass spectroscopy assay to sensitively measure mature frataxin (reduction of which is the root cause of disease) is being developed. Biomarkers to monitor neurological disease progression include imaging, electrophysiological measures and measures of nerve function, which may be measured either in serum and/or through imaging-based technologies. Potential pharmacodynamic biomarkers include metabolic and protein biomarkers and markers of nerve damage. Cardiac imaging and serum biomarkers may reflect cardiac disease progression. Considerable progress has been made in the development of biomarkers for various contexts of use, but further work is needed in terms of larger longitudinal multisite studies, and identification of novel biomarkers for additional use cases