Abstract ID 131233Poster Board 060Objectives: Chemotherapy-related cognitive impairment (CRCI) is an adverse effect associated with both cytotoxic and targeted chemotherapy, including select tyrosine kinase inhibitors (TKIs). Based on recent clinical reports demonstrating that treatment with imatinib is associated with CRCI, we have been developing a preclinical model of imatinib-induced CRCI. In the development of this model, we noted a significant decrease in p-ERK staining in the hippocampi of mice chronically treated with imatinib. Based on these findings, we sought to identify an appropriate ex vivo or in vitro model to expand upon our in vivo findings. Based on findings with these models, we sought to determine physiologically relevant, steady-state concentrations of imatinib in murine plasma and brain by characterizing its pharmacokinetic (PK) profile.Methods: To determine if imatinib would decrease p-ERK in vitro, we utilized the SH-SY5Y cell line, a neuroblastoma cell line that we and others have successfully differentiated into a neuron-like state. We treated differentiated and undifferentiated SH-SY5Y cells with 1, 10, and 100 μM imatinib for 24 h, then we performed western blotting to determine the impact of imatinib on p-ERK (n=2). We also performed cell viability assays with imatinib-treated differentiated and undifferentiated SH-SY5Y cells and neurons isolated from mice. To characterize imatinib’s steady-state brain pharmacokinetics, we collected plasma and brain from female wild-type (WT) C57BL/6 mice at 0.25, 0.5, 1, 3, and 6 h after 7 days of receiving imatinib (100 mg/kg; p.o.) (n = 4-5/group). Concentrations of imatinib in plasma and brain were determined by a validated method based on liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameter estimates were calculated with Phoenix WinNonlin. Follow-up studies were performed by inducing p-ERK activity in neurons isolated from murine hippocampi with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of vehicle, imatinib, or U0126.Results: Imatinib was cytotoxic to neurons in vitro and ex vivo when applied at concentrations >10 μM for >24 hours. Based on these data and an unexpected concentration-dependent increase in p-ERK in differentiated SH-SY5Y cells at 100 μM, we investigated imatinib’s brain accumulation and found the maximum concentration in the brain was around 1 μM. Using 1 μM imatinib, we found that imatinib decreased TPA-induced p-ERK activity.Conclusions: In the present study, we determined that imatinib reached concentrations as high as 1 μM in the brains of WT mice. These data suggest that concentrations beyond 1 μM for 24 h may not be physiologically relevant. Ex vivo experiments utilizing isolated murine hippocampal neurons and physiologically relevant concentrations and incubation times demonstrated that imatinib decreased p-ERK activity, consistent with our in vivo data.

The Wilms' Tumor 1 ( WT1) gene is a transcription factor that is recurrently mutated or commonly overexpressed in several cancer types. In acute myeloid leukemia (AML), frequent overexpression of WT1 and poor patient outcomes associated with WT1 mutations highlight its importance in the disease; however, there are no tailored treatments for these patients. Furthermore, WT1's fundamental role as either an oncogene or tumor-suppressor remains unresolved. Here we examine the roles of both wild-type and mutant WT1 in AML through epigenetic and mechanistic studies. Using our findings we propose a personalized treatment strategy. WT1 mutations are enriched in the NPM1 mut subset of AML; thus we first focused our study on a cohort of 581 patients with de novo AML and NPM1 mutations enrolled on Alliance for Clinical Trials in Oncology studies. Transcriptomic analyses revealed that WT1 mut patientsphenocopied a distinct, aberrant gene expression signature associated with FLT3 internal tandem duplications (ITD), a mutation known to activate STAT signaling and associated with poor outcome in AML. We observed that WT1 expression levels were remarkably elevated in FLT3-ITD patients and were driven by STAT5A binding to the WT1 promoter. STAT5A binding and subsequent WT1 upregulation were blocked by small molecule FLT3 inhibitors. These findings linking FLT3 activity with WT1 expression raise two possibilities: either WT1 is an oncogene cooperating with the FLT3-STAT pathway, or WT1 naturally functions to suppress FLT3 signaling in a negative feedback loop subverted by WT1 mutations. As WT1 interacts with TET2 to facilitate epigenetic remodeling and DNA binding, we performed genome-wide DNA methylation analysis on WT1 mut AML patients and observed selective hypermethylation of WT1 binding motifs consistent with loss of function. We confirmed that WT1 mutations in AML commonly cause truncation of the C-terminal DNA binding domain in our patient cohort. Using co-immunoprecipitation, ChIP-sequencing and luciferase reporter assays, we found that mutant WT1 functions as a dominant-negative, inhibiting WT1 derived from the wild-type allele. Investigation of WT1 target genes by intersecting transcriptomic and DNA methylation profiles identified hypermethylation and downregulation of miR-193a, leading to a significant downregulation of miR-193a-3p in WT1 mut AML patients. Overexpression of wild-type, but not mutated, WT1 rescued miR-193a expression in AML cell lines. Enforced expression of miR-193a significantly delayed AML onset in vivo. In addition, overexpression of miR-193a in multiple human AML cell lines and primary patient samples impaired AML cell growth and colony-forming capacity while promoting monocytic differentiation, underscoring its role as potent tumor suppressor. Therapeutic modulation of miRNA levels in cancer patients has been limited by inefficient delivery and tissue enrichment. To overcome this, we tested a novel lipid-nanoparticle (LNP) formulation of miR-193a-3p (INT-1B3), currently being investigated in a phase I clinical trial (NCT04675996). Biweekly i.v. treatments of INT-1B3 in the immunocompetent Hoxa9/Meis1 (H9M)-transduced model system prevented AML formation, highlighting the potent anti-leukemic activity of this miRNA based therapeutic. Overexpression of miR-193a-loaded LNPs downregulated FLT3 expression and suppressed STAT signaling in primary AML samples. Finally, treatment of primary AML cells with FLT3 inhibitors revealed enhanced sensitivity of WT1 mut cells in the absence of FLT3-ITD, highlighting the role of wild-type FLT3 in WT1 mut cells. In summary, we uncovered a critical negative-feedback loop maintained by WT1 to suppress FLT3 activity. Loss-of-function WT1 mutations subvert the tumor suppressor function of WT1 via failure to maintain miR-193a expression, leading to increased FLT3 expression and STAT5 signaling, subsequently impairing differentiation, increasing proliferation and disease aggressiveness in AML (Figure 1). Our findings advocate for use of FLT3 inhibition and miR-193a supplementation for treatment of WT1 mut patients, a subgroup with poor outcomes and no targeted treatment options.

ID 20840 Poster Board 250 Introduction: We performed single-cell RNA sequencing on samples from Acute Myeloid Leukemia (AML) patients with FLT3 mutations (FLT3+) pre- and post-treatment with the FLT3 inhibitor gilteritinib and identified in unresponsive patients: (i) upregulation of bone marrow (BM)-derived inflammatory cytokines, previously shown to promote disease progression and relapse in AML; and (ii) elevated expression of bone marrow kinase on chromosome X (BMX), a non-receptor tyrosine kinase from the Tec family of kinases. In FLT3+ cell lines and primary AML samples, we demonstrated that BMX gene knockout (KO) and inhibition reduced cytokine secretion. Mechanistic studies revealed that BMX kinase promotes AML cell-autonomous gilteritinib resistance through bypass signaling. Given the novel role of BMX in the cytokine network in vitro, we sought to develop a mouse model to evaluate AML cell/BM niche cell interactions in vivo to examine the mechanisms of BMX in promoting gilteritinib resistance. Methods: Genetic engineering was utilized to create a whole-body Bmx KO mouse model. To generate a syngeneic transplant model of murine Flt3+ AML, splenocytes (250,000 cells) from a female double mutant Npm1cA/+/Flt3ITD knock-in mouse (CD45.2) that produces spontaneous AML, were injected by tail vein injection (TVI) into male Bmx WT and KO mice. Starting 7 days after TVI, cohorts of mice were treated with vehicle or gilteritinib 30 mg/kg orally once daily (n=4-5 mice per cohort/per Bmx genotype). At survival endpoint, spleens were harvested and analyzed for CD45.2 cells (from vehicle-treated mice), gated for AML phenotypic markers CD11b and CD117 by flow cytometry, and murine cytokines were measured in plasma and spleen lysates by Luminex multiplex assay (from n=3-4 mice per cohort). Results: In our syngeneic transplant model of murine Flt3+ AML, gilteritinib treatment resulted in a 24-day median survival advantage over vehicle treatment (52 vs. 27.5 days) in Bmx WT mice, indicating this murine model represents a clinically relevant model of human FLT3+ AML that responds to a FLT3 inhibitor. However, no difference in survival was observed between Bmx KO and WT mice treated with gilteritinib. This is consistent with our published data showing an AML cell-autonomous role for BMX in gilteritinib resistance and suggests co-treatment with another drug to inhibit BMX or downstream mediators will be required. At study endpoint, Flt3+ AML cells were confirmed in spleens by expression of CD11b and CD117, compared to the normal spleen. We next sought to determine if microenvironment changes were occurring in Bmx KO vs. WT mice during gilteritinib treatment by assessing cytokine in plasma and AML cells in the spleen. We observed: (i) plasma CCLs/CXCLs and growth factors increased during gilteritinib treatment in Bmx WT mice but decreased in Bmx KO mice; and (ii) CCLs/CXCLs and other cytokines decreased in the spleens from Bmx KO vs. WT mice treated with gilteritinib. Conclusion: We generated a syngeneic transplant model of murine FLT3+ AML that mimics BMX-driven cytokine changes, which will be used to examine mechanisms underlying BMX-mediated AML cell/microenvironment niche cell interactions to promote gilteritinib resistance. This model will be used to evaluate a rational drug combination of gilteritnib + BMX inhibitor with translational potential in FLT3+ AML.

Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 3-year overall survival of <5%. While inhibition of kinases involved in cell cycle regulation induces synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP-0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, Chk1/2, and other cell cycle regulators. Here, we evaluated the preclinical activity of TP-0903 in TP53 mutant AML cell lines, including a single-cell clone of MV4-11 containing a TP53 mutation (R248W), Kasumi-1 (R248Q), and HL-60 (TP 53 null). TP-0903 inhibited cell viability (IC50, 12–32 nM) and induced apoptosis at 50 nM. By immunoblot, 50 nM TP-0903 upregulated pChk1/2 and pH2AX, suggesting induction of DNA damage. The combination of TP-0903 and decitabine was additive in vitro, and in vivo significantly prolonged median survival compared to single-agent treatments in mice xenografted with HL-60 (vehicle, 46 days; decitabine, 55 days; TP-0903, 63 days; combination, 75 days) or MV4-11 (R248W) (51 days; 62 days; 81 days; 89 days) (p < 0.001). Together, these results provide scientific premise for the clinical evaluation of TP-0903 in combination with decitabine in TP53 mutant AML.

Despite the clinical benefit associated with gilteritinib in relapsed/refractory acute myeloid leukemia (AML), most patients eventually develop resistance through unknown mechanisms. To delineate the mechanistic basis of resistance to gilteritinib, we performed targeted sequencing and scRNASeq on primary FLT3-ITD-mutated AML samples. Co-occurring mutations in RAS pathway genes were the most common genetic abnormalities, and unresponsiveness to gilteritinib was associated with increased expression of bone marrow-derived hematopoietic cytokines and chemokines. In particular, we found elevated expression of the TEK-family kinase, BMX, in gilteritinib-unresponsive patients pre- and post-treatment. BMX contributed to gilteritinib resistance in FLT3-mutant cell lines in a hypoxia-dependent manner by promoting pSTAT5 signaling, and these phenotypes could be reversed with pharmacological inhibition and genetic knockout. We also observed that inhibition of BMX in primary FLT3-mutated AML samples decreased chemokine secretion and enhanced the activity of gilteritinib. Collectively, these findings indicate a crucial role for microenvironment-mediated factors modulated by BMX in the escape from targeted therapy and have implications for the development of novel therapeutic interventions to restore sensitivity to gilteritinib.

The FASEB JournalVolume 35, Issue S1 PharmacologyFree Access Preclinical Characterization of TP-0903, a Novel Multikinase Inhibitor, in TP53 Mutant Acute Myeloid Leukemia Eric Eisenmann, Eric Eisenmann Divisions of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorSydney Fobare, Sydney Fobare Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorKevin Huang, Kevin Huang Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorJae Yoon Jeon, Jae Yoon Jeon Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorRobert Weber, Robert Weber Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorJosie Silvaroli, Josie Silvaroli Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorBill Larsen, Bill Larsen Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorJack Stromatt, Jack Stromatt Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorDaelynn Buelow, Daelynn Buelow Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorShelley Orwick, Shelley Orwick Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorErin Hertlein, Erin Hertlein Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorJohn Byrd, John Byrd Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorSharyn Baker, Sharyn Baker Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this author Eric Eisenmann, Eric Eisenmann Divisions of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorSydney Fobare, Sydney Fobare Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorKevin Huang, Kevin Huang Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorJae Yoon Jeon, Jae Yoon Jeon Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorRobert Weber, Robert Weber Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorJosie Silvaroli, Josie Silvaroli Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorBill Larsen, Bill Larsen Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorJack Stromatt, Jack Stromatt Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorDaelynn Buelow, Daelynn Buelow Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this authorShelley Orwick, Shelley Orwick Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorErin Hertlein, Erin Hertlein Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorJohn Byrd, John Byrd Division of Hematology, The Ohio State University, Columbus, OHSearch for more papers by this authorSharyn Baker, Sharyn Baker Division of Pharmaceutics and Pharmacology, The Ohio State University, Columbus, OHSearch for more papers by this author First published: 14 May 2021 https://doi.org/10.1096/fasebj.2021.35.S1.02109 This work was supported by funds from The Ohio State University Comprehensive Cancer Center Pelotonia Foundation (SDB) and the Pelotonia Fellowship Program (EDE). About ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Objective Acute myeloid leukemia (AML) with mutations in the tumor suppressor gene TP53 confers a dismal prognosis with 1-year overall survival of <5%. Effective treatment options are limited and current standard-of-care includes the hypomethylating agents (HMA) decitabine and azacytidine. While inhibition of kinases involved in cell cycle regulation has been shown to induce synthetic lethality in a variety of TP53 mutant cancers, this strategy has not been evaluated in mutant TP53 AML. Previously, we demonstrated that TP-0903 is a novel multikinase inhibitor with low nM activity against AURKA/B, CHEK1/2, and other cell cycle regulators (Jeon JY et al. JCI Insight 2020), thus providing scientific rationale to evaluate TP-0903 activity in TP53 mutant AML. Methods To generate an in vitro model of TP53 mutant AML, we isolated single-cell clones containing mutant (R248W) or wild-type (WT) TP53 from the established MV4-11 AML cell line; regulation of p53 targets (MDM2, p21) following gamma irradiation and inhibition of p-AURKA and p-CHEK1 by TP-0903 were assessed by immunoblot. Using these and additional TP53 mutant AML cell lines (Kasumi-1, HL-60), in vitro efficacy of TP-0903 alone and in combination with HMA was assessed in viability (MTT) and apoptosis (Annexin V) assays. In vivo efficacy studies were conducted in NSG mice following intravenous injection of HL-60 or luciferase-tagged MV4-11/TP53-R248W cells. Mice (5-10 per treatment cohort) were treated with vehicle, TP-0903 (50 mg/kg orally; 5 days on/2 days off), decitabine (0.2-0.4 mg/kg i.p.; 4 days on/10 days off) or the combination. Whole body bioluminescence imaging was performed weekly and median survival was determined. Results Compared to the clone with WT TP53, we observed a lack of MDM2 and p21 induction in MV4-11/TP53-R248W cells following gamma irradiation. In vitro, TP-0903 inhibited pAURKA and pCHEK1 at 50 nM, inhibited cell viability (IC50 values, 12-40nM), and induced apoptosis at 20-50nM. The combination of TP-0903 with HMA was additive to synergistic in all AML cell lines evaluated. In the HL-60 xenograft model, the TP-0903/decitabine combination prolonged median survival (75 days) compared to cohorts of mice treated with TP-0903 (63 days), decitabine (55 days), or vehicle (46 days) (P<0.0001). In the MV4-11/TP53-R248W xenograft model, bioluminescence imaging showed that TP-0903 alone or in combination with decitabine was more effective in suppressing the outgrowth of leukemia cells compared to mice treated with vehicle or decitabine alone (P<0.05); survival analysis is ongoing. Conclusions TP-0903 was effective in all evaluated preclinical models of TP53 mutant AML. Together, these results provide scientific premise for the initiation of a Phase 1b/2 trial of TP-0903 in combination with decitabine in TP53 mutant/complex karyotype AML under the umbrella Beat AML Master Trial. Volume35, IssueS1Special Issue: Experimental Biology 2021 Meeting AbstractsMay 2021 RelatedInformation

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity.