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ABC transporter family member genes up-regulated in Raji/253R cells

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G0–G1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)3]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253)3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253)3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177–86. ©2018 AACR.

The accumulation of phospho-ATM, γ-H2AFX and cleaved PARP in Raji compared to that in Raji/253R cells exposed to APTO-253 for 24 h.

Supplementary Table from Luxeptinib (CG-806) Targets FLT3 and Clusters of Kinases Operative in Acute Myeloid Leukemia

Chemical Synthesis of APTO-253

Supplementary Figure from Luxeptinib (CG-806) Targets FLT3 and Clusters of Kinases Operative in Acute Myeloid Leukemia

Cell growth, gene and protein expression after APTO-253 treatment. G-quadruplex binding analysis.

APTO-253 is a small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies. We sought to determine the mechanisms of action and basis for resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination studies. The cellular pharmacology of APTO-253 was analyzed in Raji lymphoma cells and a subline selected for resistance (Raji/253R). Using LC/MS/ESI analysis, APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. The intracellular content of Fe(253)3 exceeded that of the native drug by approximately 18-fold, and Fe(253)3 appears to be the most active form. Treatment of cells with APTO-253 caused DNA damage, which led us to ask whether cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug. It was found that loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 of a magnitude similar to the effects of PARP inhibitors, olaparib. Raji cells selected for 16-fold acquired resistance had 16-fold reduced accumulation of Fe(253)3. RNA-seq analysis revealed that overexpression of the ABCG2 drug efflux pump is a key mechanism of resistance. ABCG2-overexpressed HEK-293 cells were resistant to APTO-253, and inhibition of ABCG2 reversed resistance to APTO-253 in Raji/253R. APTO-253 joins the limited repertoire of drugs that can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. Mol Cancer Ther; 17(6); 1167–76. ©2018 AACR.

Supplementary Figure 6 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure 3 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure 10 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure 7 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure 4 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure Legends 1-10 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure 8 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

Supplementary Figure 5 from The Internalization and Degradation of Human Copper Transporter 1 following Cisplatin Exposure

The human copper transporter 1 (hCTR1), the major transporter responsible for copper influx, mediates one component of the cellular accumulation of cisplatin (DDP). Both copper and DDP cause rapid down-regulation of hCTR1 expression in human ovarian carcinoma cells. In this study, we investigated the mechanism of this effect using digital deconvolution microscopy and Western blot analysis of cells stained with antibodies directed at both ends of the protein. Treatment of 2008 cells with DDP in combination with inhibitors of various endosomal pathways (amiloride, cytochalasin D, nystatin, and methyl-β-cyclodextrin) showed that hCTR1 degradation was blocked by amiloride and cytochalasin D, indicating that hCTR1 was internalized primarily by macropinocytosis. Expression of transdominant-negative forms of dynamin I and Rac showed that loss of hCTR1 was not dependent on pathways regulated by either of these proteins. DDP-induced loss of hCTR1 was blocked by the proteasome inhibitors lactacystin, proteasome inhibitor 1, and MG132. This study confirms that DDP triggers the rapid loss of hCTR1 from ovarian carcinoma cells at clinically relevant concentrations. The results indicate that DDP-induced loss of hCTR1 involves internalization from the plasma membrane by macropinocytosis followed by proteasomal degradation. Because hCTR1 is a major determinant of early DDP uptake, prevention of its degradation offers a potential approach to enhancing tumor sensitivity. (Cancer Res 2006; 66(22): 10944-52)

Luxeptinib (CG-806) simultaneously targets FLT3 and select other kinase pathways operative in myeloid malignancies. We investigated the range of kinases it inhibits, its cytotoxicity landscape ex vivo with acute myeloid leukemia (AML) patient samples, and its efficacy in xenograft models. Luxeptinib inhibits wild-type (WT) and many of the clinically relevant mutant forms of FLT3 at low nanomolar concentrations. It is a more potent inhibitor of the activity of FLT3—internal tandem duplication, FLT3 kinase domain and gatekeeper mutants than against WT FLT3. Broad kinase screens disclosed that it also inhibits other kinases that can drive oncogenic signaling and rescue pathways, but spares kinases known to be associated with clinical toxicity. In vitro profiling of luxeptinib against 186 AML fresh patient samples demonstrated greater potency relative to other FLT3 inhibitors, including cases with mutations in FLT3, isocitrate dehydrogenase-1/2, ASXL1, NPM1, SRSF2, TP53, or RAS, and activity was documented in a xenograft AML model. Luxeptinib administered continuously orally every 12 hours at a dose that yielded a mean Cmin plasma concentration of 1.0 ± 0.3 μmol/L (SEM) demonstrated strong antitumor activity but no myelosuppression or evidence of tissue damage in mice or dogs in acute toxicology studies. On the basis of these studies, luxeptinib was advanced into a phase I trial for patients with AML and myelodysplastic/myeloproliferative neoplasms.

The molecular underpinnings of acquired resistance to carboplatin are poorly understood and often inconsistent between in vitro modeling studies. After sequential treatment cycles, multiple isogenic clones reached similar levels of resistance, but significant transcriptional heterogeneity. Gene-expression based virtual synchronization of 26,772 single cells from 2 treatment steps and 4 resistant clones was used to evaluate the activity of Hallmark gene sets in proliferative (P) and quiescent (Q) phases. Two behaviors were associated with resistance: (1) broad repression in the P phase observed in all clones in early resistant steps and (2) prevalent induction in Q phase observed in the late treatment step of one clone. Furthermore, the induction of IFNα response in P phase or Wnt-signaling in Q phase were observed in distinct resistant clones. These observations suggest a model of resistance hysteresis, where functional alterations of the P and Q phase states affect the dynamics of the successive transitions between drug exposure and recovery, and prompts for a precise monitoring of single-cell states to develop more effective schedules for, or combination of, chemotherapy treatments.

LGR5 and LGR6 mark epithelial stem cells in many niches including the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. Human ovarian cancers express these receptors at high levels and express one of their ligands, RSPO1, at levels uniquely higher than all other tumor types except mesothelioma. Reasoning that these receptors are also important to tumor stem cells, arming the LGR binding domain of RSPO1 with a cytotoxin may permit depletion of the tumor stem cells. The Fu1-Fu2 receptor binding domain of RSPO1 (R1FF), containing a sortase recognition sequence at the C-terminal end, was produced in bacteria and a single molecule of MMAE was attached to each R1FF through a val-cit-PAB linker using the sortase reaction, thus producing a homogeneous population of armed molecules. R1FF-MMAE demonstrated (1) selective LGR-dependent binding, uptake, and cytotoxicity; (2) low nM cytotoxicity to multiple types of human tumor cell lines in vitro; (3) favorable plasma pharmacokinetic properties when administered iv with an elimination half-life of 27.8 h; (4) favorable absorption from the peritoneal cavity; and (5) therapeutic activity in aggressive xenograft models of ovarian cancer in the absence of any weight loss or other adverse events. These results demonstrate that the Fu1-Fu2 domain of RSPO1 can be exploited to deliver a potent cytotoxin to tumor cells that express the LGR4-6 family of stem cell receptors.

Background/aim R-spondins control WNT signaling and RSPO1 and LGR6, two of its receptors, are uniquely expressed at high levels in high-grade serous ovarian cancer (HGSOC). The aim of this study was to assess the interrelations between the expression of the RSPOs and LGRs in HGSOC and in the ovarian surface (OSE) and fallopian tube surface epithelium (FTSE) from which HGSOC arises. Materials and methods Analysis of TCGA (HGSOC), CCLE (ovary), and other publicly accessed RNA-Seq data using UC San Diego Computational Cancer Analysis Library (CCAL) to perform differential expression analysis, association studies, and gene set inspection using the single-sample GSEA method. Additionally, we employed multiple publicly available databases including StringDB, Human Protein Atlas, and cBioPortal to aid the investigation. Results Among normal tissues, expression of RSPO1, LGR5 and LGR6 was highest in the fallopian tube. The relative levels of expression of the RSPOs and LGRs in the OSE and FTSE matched those in HGSOC. RSPO1 and LGR6 were highly co-expressed in all three tissues. Gene set enrichment analysis (GSEA) showed that expression of RSPO1 was strongly linked to the enrichment of three separate WNT-driven GO pathways. Analysis of genes that impacted overall survival identified two other immediately adjacent genes that control WNT signaling, KREMEN1 and ZNRF3 whose expression and copy number were coordinately linked. Conclusion RSPO1 and LGR6 are coordinately expressed in HGSOC and the two normal tissues from which this tumor arises, and their expression is linked to WNT signaling pathways known the control cell fate and proliferation.

In nanomedicine, determining the spatial distribution of particles and drugs, together and apart, at high resolution within tissues, remains a major challenge because each must have a different label or detectable feature that can be observed with high sensitivity and resolution. We prepared nanoparticles capable of enzyme-directed assembly of particle therapeutics (EDAPT), containing an analogue of the Pt(II)-containing drug oxaliplatin, an 15N-labeled monomer in the hydrophobic block of the backbone of the polymer, the near-infrared dye Cy5.5, and a peptide that is a substrate for tumor metalloproteinases in the hydrophilic block. When these particles reach an environment rich in tumor associated proteases, the hydrophilic peptide substrate is cleaved, causing the particles to accumulate through a morphology transition, locking them in the tumor extracellular matrix. To evaluate the distribution of drug and EDAPT carrier in vivo, the localization of the isotopically labeled polymer backbone was compared to that of Pt by nanoscale secondary ion mass spectrometry (NanoSIMS). The correlation of NanoSIMS with super-resolution fluorescence microscopy revealed the release of the drug from the nanocarrier and colocalization with cellular DNA within tumor tissue. The results confirmed the dependence of particle accumulation and Pt(II) drug delivery on the presence of a Matrix Metalloproteinase (MMP) substrate and demonstrated antitumor activity. We conclude that these techniques are powerful for the elucidation of the localization of cargo and carrier, and enable a high-resolution assessment of their performance following in vivo delivery., Multiscale imaging methods to track drug-loaded nanoparticles are needed. Correlated optical and isotopic nanoscopy were used to study Pt(II)-loaded enzyme responsive micelle distribution in tissues.

INTRODUCTION: APTO-253 represses expression of the MYC oncogene by targeting a conserved G-quadruplex structure in its promoter, down-regulates MYC mRNA and protein levels and induces apoptosis in AML cell lines and marrow samples from patients with AML, MDS, and MPN in vitro. After injection, a large fraction of APTO-253 binds iron and transforms to the Fe(253) 3 complex which retains full activity. APTO-253 has been granted orphan drug designation for AML by the US FDA and is being studied in a Phase 1a/b clinical trial in patients with relapsed or refractory AML (R/R AML) or high-risk myelodysplasias (high-risk MDS) (NCT02267863). AIMS: Primary objectives are to determine the safety and tolerability of APTO-253, MTD, dose limiting toxicities (DLT), and the RP2D. Key secondary objectives are to assess the pharmacokinetic (PK) profile, pharmacodynamic (PD) activity, and preliminary evidence of antitumor activity. METHODS: Eligible patients have R/R AML or high-risk MDS for which either standard treatment has failed, is no longer effective, or can no longer be administered safely. Treatment- emergent adverse events (TEAEs) and tumor responses are evaluated using International Working Group criteria. APTO-253 is administered by IV infusion once weekly on days 1, 8, 15, and 22 of each 28-day cycle; ascending dose cohorts were enrolled at a starting dose of 20 mg/m 2 with planned escalation to 403 mg/m 2. RESULTS: As of June 7, 2021, a total of 18 patients (median age 64.0 years, 16 AML and 2 high-risk MDS) with a median of 2.5 prior treatments (range of 1 - 9) have been treated with APTO-253 at doses of 20 (n=1), 40 (n=1), 66 (n=4), 100 (n=4) and 150 mg/m 2 (n=8). Most patients were RBC (87.5% of AML and 100% of MDS) and/or platelet (75% of AML and 50% MDS) transfusion-dependent. No DLTs or drug-related serious adverse events have been reported. Only 1 patient had a drug-related TEAE of grade 3 or greater (fatigue, Grade 3, probably related). Preliminary PK analysis (Figure 1) showed that serum levels of APTO-253 were dose proportional. C max and AUC 0-72h for C1D1 dosing were 0.06, 0.02, 0.36 ± 0.37, 0.44 ± 0.41 and 0.72 ± 0.70 µM and 0.11, 0.15, 3.98 ± 1.77, 4.79 ± 0.87 and 2.51 ± 1.73 µM*h for dose levels of 20, 40, 66, 100 and 150 mg/m 2, respectively. Plasma levels for Fe(253) 3 were significantly higher than those for the APTO-253 monomer. For example, C max and AUC 0-72h of Fe(253) 3 for C1D1 dosing of patients in Cohort 150 mg/m 2 were 2- and 20- fold higher than the ATPO-253 monomer at 15.09 ± 0.42 µM and 51.52 ± 28.26 µM*h, respectively. Following dosing at 150 mg/m 2, serum concentrations of Fe(253) 3 were above 0.5 µM for > 48 h, which approaches the therapeutic range based on in vitro studies. CONCLUSIONS: APTO-253 has been well-tolerated at doses of 20, 40, 66, 100 and 150 mg/m 2 over multiple cycles and escalated to 210 mg/m 2 (Cohort 6). PK analysis revealed that APTO-253 is rapidly transformed to and co-exists with the Fe(253) 3 in serum from R/R AML and high-risk MDS patients. Enrollment of patients at the 210 mg/m 2 dose level is ongoing and updated clinical data will be presented at the meeting. Figure 1 Figure 1. Disclosures Arellano: KITE Pharma, Inc: Consultancy; Syndax Pharmaceuticals, Inc: Consultancy. Levy: AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Other: Promotional speaker; Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; Morphosys: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; Epizyme: Consultancy, Other: Promotional speaker; Takeda: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; Dova: Consultancy, Other: Promotional speaker; Novartis: Consultancy, Other: Promotional speaker; TG Therapeutics: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau; Gilead Sciences, Inc.: Consultancy, Honoraria, Speakers Bureau; Beigene: Consultancy, Honoraria, Speakers Bureau; Amgen Inc.: Consultancy, Honoraria, Other: Promotional speaker, Speakers Bureau. Mahadevan: caris: Speakers Bureau; Guardanthealt: Speakers Bureau; PFIZER: Other: Clinical trial Adverse events committee; TG Therapeuticals: Other: Clinical trial Adverse events committee. Zhang: Aptose Biosciences, Inc.: Current Employment. Rastgoo: Aptose Biosciences, Inc.: Current Employment. Jin: Aptose Biosciences, Inc.: Current Employment. Marango: Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Howell: Aptose Biosciences, Inc.: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rice: Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties; Oncolytics Biotech Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Bejar: Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company; Takeda: Research Funding; BMS: Consultancy, Research Funding; Gilead: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Astex: Consultancy; Silence Therapeutics: Consultancy.

INTRODUCTION: Luxeptinib (CG-806) is a potent, non-covalent oral inhibitor of BTK and FLT3. It suppresses BCR signaling pathways (LYN, SYK, BTK, AKT, ERK) and other oncogenic pathways in cell lines and primary CLL cells, kills malignant B-cells insensitive to ibrutinib or venetoclax at low nM concentrations, and shows enhanced activity in combination with venetoclax. Luxeptinib is currently being evaluated in a Phase 1a/b trial in patients with relapsed or refractory B-cell malignancies (NCT03893682). AIMS: Primary objectives are to assess the safety and tolerability of luxeptinib and determine the recommended expansion dose for future clinical trials in patients with R/R CLL/SLL or B-NHL. Key secondary objectives include elucidation of the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics, and evidence of antitumor activity. METHODS: Luxeptinib is administered continuously as oral capsules BID in 28-day cycles, in ascending cohorts of 3 patients. Treatment emergent adverse events (TEAEs) and tumor responses are evaluated per disease specific guidelines. RESULTS: As of June 7, 2021, a total of 23 patients (median age 64.0 years, 11 CLL/SLL, 4 follicular lymphoma (FL), 1 Richter's transformation lymphoma, 2 Waldenstrom's macroglobulinemia (WM), 2 mantle cell lymphoma, 3 diffuse large B cell lymphoma (DLBCL)) with a median of 3 prior systemic regimens (range of 1 - 12) have been treated with luxeptinib at doses of 150 mg (n=1), 300 mg (n=1), 450 mg (n=9), 600 mg (n=4) and 750 mg (n=8). Drug related grade ≥ 3 TEAEs included neutropenia (n=5, 21.7%), leukocytosis (n=2, 8.7%), decreased white blood cell count, decreased platelet count, anemia, increased alanine aminotransferase, diarrhea, hypertension, and headache (n=1, 4.3 % each). One patient, who experienced progressive hypertension before receiving luxeptinib, developed grade 1 hypertension during screening evolving to grade 2 prior to dosing on C1D1, experienced grade 4 hypertension after luxeptinib treatment at 750 mg BID for 5 days. While this was considered a DLT per protocol, luxeptinib plasma levels in this patient were lower than those observed in patients at this and lower dose levels, and no drug-related hypertension has been observed in any other patient treated to date. The average steady-state (C min) plasma levels of luxeptinib in patients treated with 750 mg BID were > 1µM and were sustained over multiple cycles. PD analysis demonstrated that C min of luxeptinib in the plasma of patients treated with doses of 300 mg or higher significantly reduced levels of phosphorylated BTK, SYK, ERK and FLT3 in EOL-1 reporter cells in a plasma inhibitor activity assay, demonstrating multi-target engagement. Response evaluations were available for 12 patients who had at least one imaging study or IgM measurements (WM patients) after starting treatment. Eight out of these 12 patients had various reductions of lesion size or IgM measurements compared to baseline (3 SLL, 2 FL, 1 CLL, 1 WM and 1 DLBCL), demonstrating anti-tumor activity. FDG PET-CT scans in one FL patient, who had 2 prior regimens, revealed lesion growth during treatment with luxeptinib at 450 mg BID for 7 cycles. After dose-escalation to 600 mg BID in Cycle 8, lesion measurements demonstrated continuous reduction. By C15D1, target lesions decreased by 42.5% and 11.3% when compared with peak tumor size and baseline measurements, respectively. One WM patient in Cohort 5 (750 mg BID), who had 2 prior regimens including ibrutinib, has had a 24.9% reduction in IgM level by C3D1 compared with baseline as her best response so far. CONCLUSIONS: Luxeptinib has a favorable safety profile in patients treated with 150 mg to 600 mg BID over multiple cycles. A single apparent DLT (grade 4 hypertension in one patient with pre-existing hypertension) led to expansion of the 750 mg dose level to 6 patients. Pharmacodynamic studies documented inhibition of SYK, BTK and ERK in the BCR signaling pathway. Anti-tumor activity was observed in multiple B-NHL subtypes and CLL/SLL patients. Enrollment of patients with R/R CLL/SLL and B-NHL at dose level 5 (750 mg) is ongoing and updated clinical data will be presented at the meeting. Disclosures Samaniego: Arog: Research Funding; Imbrium: Membership on an entity's Board of Directors or advisory committees. Burke: AstraZeneca: Consultancy; Verastem: Consultancy; Bristol Myers Squibb: Consultancy; Epizyme: Consultancy; Kura: Consultancy; Kymera: Consultancy; SeaGen: Consultancy, Speakers Bureau; X4 Pharmaceuticals: Consultancy; Adaptive Biotechnologies: Consultancy; Beigene: Consultancy, Speakers Bureau; AbbVie: Consultancy; MorphoSys: Consultancy; Roche/Genentech: Consultancy. Mahadevan: caris: Speakers Bureau; Guardanthealt: Speakers Bureau; PFIZER: Other: Clinical trial Adverse events committee; TG Therapeuticals: Other: Clinical trial Adverse events committee. Cherry: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sadiq: Dava Oncology: Honoraria. Reid: ADC Therapeutics: Other: Serves as Principle Investigator, Research Funding; Aptose Biosciences: Other: Serves as Principle Investigator, Research Funding; Millennium Pharmaceuticals: Other: Serves as Principle Investigator, Research Funding; Xencor: Other: Serves as Principle Investigator, Research Funding. Roeker: TG Therapeutics: Consultancy; Pfizer: Consultancy, Research Funding; AbbVie, AstraZeneca, Janssen, LOXO, Pharmacyclics, TG Therapeutics, Vaniam Group, Verastem: Consultancy; Loxo Oncology: Consultancy; Abbot Laboratories: Current equity holder in publicly-traded company; Pharmacyclics: Consultancy. Melear: Astrazeneca: Speakers Bureau; TG Therapeutics: Speakers Bureau; Janssen: Speakers Bureau. Zhang: Aptose Biosciences, Inc.: Current Employment. Rastgoo: Aptose Biosciences, Inc.: Current Employment. Benbatoul: Aptose Biosciences, Inc.: Current Employment. Haney: Aptose Biosciences, Inc.: Current Employment. Jin: Aptose Biosciences, Inc.: Current Employment. Marango: Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Howell: Aptose Biosciences, Inc.: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rice: Oncolytics Biotech Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Bejar: Astex: Consultancy; Silence Therapeutics: Consultancy; Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company; Epizyme: Consultancy, Honoraria; Takeda: Research Funding; Gilead: Consultancy, Honoraria; BMS: Consultancy, Research Funding.

INTRODUCTION: Luxeptinib (CG-806) is a potent oral small molecule inhibitor of the wild type and all mutant forms of the FLT3 kinase, including ITD, D835Y, and F691L. Luxeptinib simultaneously suppresses additional signaling pathways in AML cells (CSF1R, PDGFRα, TRK, SYK, BTK, LYN, AKT, ERK, MAPK), kills primary AML cells insensitive to other FLT3 inhibitors at pM and low nM concentrations, and shows enhanced activity in combination with venetoclax. Patient-derived AML cells retain sensitivity to luxeptinib even when harboring mutations of NPM1, IDH1, ASXL1, or TP53. Luxeptinib is being evaluated in a Phase 1a/b trial in patients with relapsed or refractory (R/R) AML (NCT04477291). AIMS: The primary objectives are to assess the safety and tolerability of luxeptinib and to determine the recommended expansion dose for future clinical trials in patients with R/R AML. Key secondary objectives include elucidation of the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics, and evidence of antitumor activity. METHODS: The study is enrolling patients with relapsed or refractory de novo AML, secondary AML, or therapy-related AML. Luxeptinib is administered continuously as oral capsules BID in 28-day cycles, in ascending cohorts of 3 or 4 patients. Treatment emergent adverse events (TEAEs) and tumor responses are evaluated using CTCAE and European Leukemia Net criteria, respectively. RESULTS: As of June 7, 2021, a total of 10 patients (median age 74.5 years, 6 (60%) FLT3-ITD, and 4 (40%) FLT3-WT per local labs) with a median of 3 prior treatments (range, 1 - 8) have been treated with luxeptinib at dose levels of 450 mg BID (n=6) and 600 mg BID (n=4). Majority of patients (≥ 60 %) were RBC and / or platelet transfusion dependent. Drug related TEAEs at Grade ≥ 3 included decreased platelet count, anemia, and pericardial effusion (n=1, 10% each). One patient, who received 450 mg BID (Cohort 1), had Grade 3 pericardial effusion, was assessed initially as a DLT possibly related to luxeptinib and led to expansion of Cohort 1 to treat 6 patients. No DLT was observed in the other 5 patients in Cohort 1 and supported dose escalation to 600 mg BID (Cohort 2), where no DLT was observed in 4 patients and escalated to 750 mg BID (Cohort 3). The steady-state (C min) plasma levels of luxeptinib in the patients treated with 600 mg BID were 0.6 - 1.6 µM by the end of Cycle 1. PD analysis demonstrated luxeptinib in the plasma of patients reduced FLT3 phosphorylation > 85% at concentrations > 0.2 µM while significantly reducing phosphorylation of ERK, SYK and PDGFRα in MOLM14 and / or EOL-1 reporter cells in a plasma inhibitor activity assay, demonstrating target engagement by luxeptinib. Disease evaluation via flow cytometry, PCR-based FLT3 mutation tests and targeted exome sequencing on bone marrow and peripheral blood revealed anti-leukemic activity of luxeptinib in 3 relapsed AML patients with FLT3-ITD mutations in Cohort 1. One heavily pretreated AML patient (8 prior regimens including alloSCT and FLT3 inhibitors gilteritinib and crenolanib) had 99% reduction of blasts in peripheral blood (from 6.38x10 3/µL at C1D1 to 0.09x10 3/µL at C1D15), though the decrease in blasts reversed during Cycle 2. Another AML patient (2 prior regimens) had FLT3-ITD VAF 0.62 at screening, which decreased 80% to 0.12 by C4D9. This patient had simultaneous reductions of clones with mutations of GATA2 R337K, TET2 R1359C, SRSF2 P95L and ASXL1 E635R, whereas a PTPN11 mutant clone emerged. MRD-negative complete remission (CR) was confirmed in one FLT3-ITD AML patient (6 prior regimens including 2 alloSCT and FLT3 inhibitor sorafenib) at Cycle 5 of treatment evidenced by reduction of FLT3-ITD VAF and blasts in bone marrow to below limit of detection (LOD) of PCR-based FLT3-ITD assay and high-sensitivity flow cytometry (LOD < 0.1%), respectively. CONCLUSIONS: As of June 7, 2021, luxeptinib is well tolerated at dose levels of 450 and 600 mg BID over multiple cycles and escalated to 750 mg BID (Cohort 3). Pharmacodynamic studies documented inhibition of FLT3 signaling, and anti-leukemic activity has been observed in heavily pretreated relapsed FLT3-ITD AML patients as evidenced by significant reduction of FLT3-ITD VAF and blasts in bone marrow and / or peripheral blood. One FLT3-ITD AML patient has had confirmed MRD negative CR and continues treatment. Enrollment of patients with R/R AML in Cohort 3 is ongoing and updated clinical data will be presented at the meeting. Disclosures Goldberg: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celularity: Research Funding; DAVA Oncology: Honoraria; Arog: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Prelude Therapeutics: Research Funding; Aptose: Consultancy, Research Funding; Aprea: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Koller: Novartis: Consultancy. Altman: Amgen: Research Funding; Astellas: Consultancy, Other: Advisory Board, Research Funding; Kura Oncology: Consultancy; Biosight: Consultancy, Other: Travel fees, Research Funding; Kura: Research Funding; GlycoMimetics: Other: Participation on an advisory board; Daiichi Sankyo: Consultancy; ALZ Oncology: Research Funding; Syros: Consultancy; Theradex: Consultancy, Other: Advisory boards; Kartos: Research Funding; AbbVie: Consultancy, Other: Advisory Board, Research Funding; Aprea: Research Funding; BMS: Research Funding; Boehringer Ingelheim: Research Funding; Fujifilm: Research Funding; Immunogen: Research Funding. Cherry: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zhang: Aptose Biosciences, Inc.: Current Employment. Rastgoo: Aptose Biosciences, Inc.: Current Employment. Benbatoul: Aptose Biosciences, Inc.: Current Employment. Jin: Aptose Biosciences, Inc.: Current Employment. Su: Aptose Biosciences, Inc.: Current Employment. Marango: Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Howell: Aptose Biosciences, Inc.: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rice: Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties; Oncolytics Biotech Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Bejar: Astex: Consultancy; Epizyme: Consultancy, Honoraria; Silence Therapeutics: Consultancy; BMS: Consultancy, Research Funding; Gilead: Consultancy, Honoraria; Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company; Takeda: Research Funding.

INTRODUCTION: APTO-253 is the only known small molecule in clinical development to target a conserved G-quadruplex structure in the promoter of the MYC oncogene and interrupt MYC gene expression. In preclinical studies of acute myeloid leukemia (AML) cell lines, APTO-253 potently down-regulates MYC gene expression, reduces MYC mRNA and protein level, depletes cells of the MYC oncoprotein and induces apoptosis (Local et al., 2018). APTO-253 demonstrated broad killing of primary mononuclear cells isolated from the bone marrow of patients with AML, MDS, or MPN and enhanced AML cell killing when combined with BET bromodomain inhibitors or FLT3 inhibitors (Kurtz, et al., 2017). Fe(253)3, a ferrous complex formed from parental APTO-253 when it chelates iron, exhibits similar in vitro anti-tumor potency as its monomeric form (Tsai, et al., 2018). Because dysregulated MYC is considered a major oncogenic operator in AML and myelodysplasias, and because APTO-253 has such a distinct cytotoxic mechanism, APTO-253 has been granted orphan drug designation for the treatment of AML by the US FDA and is currently in a Phase 1a/b clinical trial (NCT02267863) in patients with relapsed or refractory AML (R/R AML) or high-risk myelodysplasias (high-risk MDS). AIMS: Primary objectives are to determine the safety and tolerability of APTO-253, to determine the maximum tolerated dose and the dose limiting toxicities (DLT), and to establish the recommended Phase 2 dose for future clinical trials in patients with R/R AML or high-risk MDS. Key secondary objectives are to assess the pharmacokinetic (PK) profile, pharmacodynamic (PD) activity, and preliminary evidence of antitumor activity. METHODS: Eligible patients are those with R/R AML or high-risk MDS for which either standard treatment has failed, is no longer effective, or can no longer be administered safely. Treatment- emergent adverse events (TEAEs) and tumor responses are evaluated using International Working Group criteria. APTO-253 is administered by IV infusion once weekly on days 1, 8, 15, and 22 of each 28-day cycle; ascending dose cohorts will enroll at a starting dose of 20 mg/m2 with planned escalating to 403 mg/m2. RESULTS: As of July 28, 2020, a total of 10 patients (age 66.1 ± 13.58 years, male 50%, female 50%, 8 AML and 2 MDS) have been treated with APTO-253 in this clinical trial at doses of 20 mg/m2 (n=1), 40 mg/m2 (n=1), 66 mg/m2 (n=4), and 100 mg/m2 (n=4). All 8 AML patients and 1 MDS patient were RBC and platelet transfusion dependent; 1 MDS patient was RBC transfusion dependent. No DLTs or drug-related serious adverse events have been reported. Possible drug related grade 2 TEAEs included fatigue, increased alkaline phosphatase, decreased appetite, hematoma, hypokalemia, thrombophlebitis, upper respiratory tract in 1 (10%) patient each. Only 1 TEAE of grade 3 or greater (fatigue, considered possibly drug-related) has occurred to date. Preliminary PK analysis showed plasma levels of APTO-253 were dose proportional. Cmax and AUC0-24h on cycle 1 day 1 (C1D1) were 0.18, 0.07, 0.28 ± 0.15 and 0.77 ± 0.63 µM and 0.08, 0.13, 1.14 ± 0.57, 1.84 ± 0.41 µM*h for dose levels of 20 mg/m2, 40 mg/m2, 66 mg/m2, and 100 mg/m2, respectively. Not surprisingly, Fe(253)3 was detected in the patients' plasma immediately after dosing and at a significantly higher concentration than the APTO-253 monomer. For example, Cmax and AUC0-24h of Fe(253)3 on C1D1 of patients in Cohort 66 mg/m2 were 3- and 8-fold higher than the monomer at 0.92 ± 0.29 µM and 20.61 ± 9.01 µM*h, respectively. The levels of MYC mRNA in the whole blood, a PD biomarker of APTO-253 and Fe(253)3 measured by RT-qPCR, were reduced 20-48% at 24 h post-dose as compared to pre-dose in the first 3 cohorts (other samples of Cohort 100 mg/m2 in process), suggesting target engagement by the drug. CONCLUSIONS: APTO-253 has been well-tolerated in patients treated with 20, 40, 66, and 100 mg/m2 over multiple cycles. PK analysis revealed APTO-253 monomer rapidly transformed to and co-existed with the Fe(253)3 complex in peripheral blood and their exposures resulted in suppression of MYC expression in whole blood samples from R/R AML and high-risk MDS patients. Enrollment of patients at the 150 mg/m2 dose level is underway and updated clinical data will be presented at the meeting. Disclosures Bejar: Aptose Biosciences, Inc: Current Employment, Current equity holder in publicly-traded company. Zhang:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Rastgoo:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Benbatoul:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Jin:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Thayer:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Sheng:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Chow:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Montalvo-Lugo:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Marango:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Howell:Aptose Biosciences, Inc.: Current equity holder in publicly-traded company. Rice:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company.

The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

INTRODUCTION: CG-806 is a potent, non-covalent oral inhibitor of Bruton's tyrosine kinase (BTK), FMS-like tyrosine kinase 3 (FLT3) and certain other validated driver kinases of malignancies, while sparing kinases typically associated with clinical toxicity. In cell lines and primary samples from chronic lymphocytic leukemia (CLL) patients, CG-806 suppressed BCR signaling (LYN, SYK, BTK, AKT, ERK) and pathways operative in AML (FLT3, AKT, CSF1R, MAPK, PDGFRα, others), killed malignant B-cell cells insensitive to ibrutinib or venetoclax at low nM concentrations, and showed enhanced activity in combination with venetoclax. CG-806 is currently being evaluated in a Phase 1 a/b trial in patients with CLL and select relapsed or refractory B-cell malignancies (NCT03893682). A parallel Phase 1 a/b clinical study of CG-806 in patients with relapsed or refractory (R/R) FLT3-mutant or FLT3-wildtype AML is in progress (NCT04477291). AIMS: The primary objectives of the clinical study NCT03893682 are to assess the safety and tolerability of CG-806 and to determine the recommended Phase 2 dose for future clinical trials in patients with advanced CLL/small lymphocytic lymphoma (SLL) or non-Hodgkin lymphoma (NHL). Key secondary objectives include elucidation of the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics, preliminary evidence of antitumor activity, and selection of a starting dose for a trial in patients with R/R AML. METHODS: Eligible patients are those with CLL/SLL or NHL for which either the standard treatment has failed, is no longer effective, or can no longer be administered safely. Treatment emergent adverse events (TEAEs) and tumor responses are evaluated per disease specific guidelines. CG-806 is administered as capsules PO BID in 28-day cycles. RESULTS: As of July 24, 2020, a total of 11 patients (age 66.2 ± 7.4 years, male 54.5%, female 45.5%, 5 CLL/SLL, 3 follicular lymphoma, 1 Richter transformation lymphoma, 1 Waldenstrom's macroglobulinemia, 1 mantle cell lymphoma) with a median of 7 prior systemic regimens have been treated with CG-806 at doses of 150 mg (n=1), 300 mg (n=1), 450 mg (n=5), and 600 mg (n=4). No dose limiting toxicities or related serious adverse event have been reported. The most common drug related or possible related TEAEs included diarrhea, nausea, neutropenia in 3 (27.3%) each; fatigue, lymphocytosis and vomiting in 2 (18.2%) patients each. Drug related or possible related grade 3 or greater TEAEs included diarrhea (n=1, 9.1%) and neutropenia (n=2, 18.2%), but no fevers were reported - those two neutropenia events occurred in a patient with Richter transformation from CLL who had failed 12 prior regimens including ibrutinib, venetoclax, binutuzumab, CAR-NK and CAR-T and was found to have a prior therapy-related myeloid neoplasm after enrollment. Among three classic CLL patients placed on study, all experienced on-target lymphocytosis upon initiation of CG-806 therapy, as well as inhibition of phospho-BTK measured in the whole blood by ELISA and measured by a plasma inhibitor activity (PIA) assay with reporter cells. Steady state plasma levels of CG-806 in patients treated with CG-806 at 300 mg or higher doses dramatically reduced phosphorylated BTK-Y551, BTK-Y223, SYK-Y525/526, ERK-T202/Y204 and FLT3-Y591 in EOL-1 reporter cells, as determined by PIA assay, documenting multi-target engagement. Preliminary PK analyses showed the steady-state (Cmin) plasma levels of CG-806 on cycle 2 day 1 in patients treated with ≥300 mg BID were generally in the 1µM range and were sustained over multiple cycles. CONCLUSIONS: CG-806 demonstrated a favorable safety profile in patients treated to date with 150, 300, 450 or 600 mg BID over multiple cycles. Oral absorption in patients produced plasma concentrations known to be effective in murine leukemia models. Pharmacodynamic studies using patient whole blood and plasma documented inhibition of SYK, BTK and ERK in the BCR signaling pathway. Further, the trial allowed selection of a potentially therapeutically active starting dose for AML patients (450mg). Enrollment of patients with R/R CLL/SLL and NHL at dose level 5 (750mg) is ongoing and updated clinical data will be presented at the meeting. Disclosures Bejar: Aptose Biosciences, Inc: Current Employment, Current equity holder in publicly-traded company. Zhang:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Rastgoo:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Benbatoul:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Jin:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Thayer:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Sheng:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Chow:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Montalvo-Lugo:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Marango:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company. Howell:Aptose Biosciences, Inc.: Current equity holder in publicly-traded company. Rice:Aptose Biosciences, Inc.: Current Employment, Current equity holder in publicly-traded company.

Single cell RNA-sequencing (scRNA-seq) has advanced studies of heterogeneous tissues and cell populations in both healthy and disease states. Dimensionality reduction methods leverage this highly granular data to identify cellular states not captured by “bulk” gene expression or to organize cells along ‘pseudotime' trajectories related to biological processes such as cell cycle or cellular differentiation. Cellular states and trajectories are often annotated using individual marker genes and not gene sets or pathways, despite their more reliable and interpretable association to biological processes. Gene Set Enrichment Analysis (GSEA) was designed to test the enrichment of gene sets in bulk transcriptomic data, but due to the sparsity of the expression data, has not been adapted to single cell analysis. We propose a method to compute gene set enrichment for scRNA-seq data, which calculates an enrichment score for each cell in the dataset. We apply this method to a set of matched ovarian cancer cell lines with acquired resistance to carboplatin. After aligning the cells to a cell-cycle derived pseudotime trajectory, the activity of pathways which were dysregulated in bulk expression profiles appeared to change with the cell cycle progression. The analysis further identified interferon alpha signaling induction in resistant cells, independent of differences in cell cycle progression. Hence, by quantifying and analyzing pathway-level transcriptional activity in the context of a single-cell pseudotime trajectory, the proposed method provides more interpretable annotation of biological processes altered in cancer progression and treatment. Citation Format: Alexander T. Wenzel, Devora Champa, Stephen B. Howell, Jill P. Mesirov, Olivier Harismendy. A gene set enrichment analysis approach in single-cells along pseudotime trajectories reveals the dynamic activity of oncogenic pathways [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 4411.

SummaryCarcinogens form adducts with the DNA which, when not properly repaired, can lead to mutations and drive oncogenesis. The identity, sequence specificity and mutagenicity of most DNA-adducts is however poorly understood and current molecular assays are limited in their scope and scalability. We present a novel genome-wide DNA adduct sequencing (Ad-Seq) assay to map the location of DNA-adducts at single-nucleotide resolution. Ad-Seq enriches for DNA fragments containing nuclease digestion resistant DNA-adducts. The genomic location of the resulting reads is aggregated in a quantitative profile showing the DNA-adduct sequence context. Ad-Seq is quantitative and confirms known specificity of damages from Ultra-Violet light (di-pyrimidine) and cisplatin (AG and GG di-purines). Furthermore, in cells, Ad-Seq profile can be compared to chromatin segments to show that cisplatin associated adducts are depleted in open and active chromatin regions. The Ad-Seq assay can therefore generate a broad DNA signature of DNA damage and, by comparing to mutagen exposure or downstream mutational profile and signatures, be used to improve our understanding of cancer molecular etiology.

APTO-253 is a small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies. We sought to determine the mechanisms of action and basis for resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination studies. The cellular pharmacology of APTO-253 was analyzed in Raji lymphoma cells and a subline selected for resistance (Raji/253R). Using LC/MS/ESI analysis, APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. The intracellular content of Fe(253)3 exceeded that of the native drug by approximately 18-fold, and Fe(253)3 appears to be the most active form. Treatment of cells with APTO-253 caused DNA damage, which led us to ask whether cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug. It was found that loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 of a magnitude similar to the effects of PARP inhibitors, olaparib. Raji cells selected for 16-fold acquired resistance had 16-fold reduced accumulation of Fe(253)3. RNA-seq analysis revealed that overexpression of the ABCG2 drug efflux pump is a key mechanism of resistance. ABCG2-overexpressed HEK-293 cells were resistant to APTO-253, and inhibition of ABCG2 reversed resistance to APTO-253 in Raji/253R. APTO-253 joins the limited repertoire of drugs that can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. Mol Cancer Ther; 17(6); 1167–76. ©2018 AACR.

Wnt signaling plays a fundamental role in patterning of the embryo and maintenance of stem cells in numerous epithelia. Epithelial stem cells are closeted in niches created by surrounding differentiated cells that express secreted Wnt and R-spondin proteins that influence proliferation rate and fate determination of stem cell daughters. R-spondins act through the LGR receptors to enhance Wnt signaling. This close association of stem cells with more differentiated regulatory cells expressing Wnt-pathway ligands is a feature replicated in all of the epithelial stem cell systems thus far examined. How the stem cell niche operates through these short-range interactions is best understood for the crypts of the gastrointestinal epithelium and skin. Less well understood are the stem cells that function in the ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE). While the cuboidal OSE appears to be made up of a single cell type, the cells of the FTE progress through a life cycle that involves differentiation into ciliated and secretory subtypes that are eventually shed into the lumen in a manner similar to the gastrointestinal epithelium. Available evidence suggests that high grade serous ovarian carcinoma (HGSOC) originates most often from stem cells in the FTE and that Wnt signaling augmented by LGR6 supports tumor development and progression. This review summarizes current information on LGR5 and LGR6 in the OSE and FTE and how their niches are organized relative to that of the gastrointestinal epithelium and skin.

Peritoneal carcinomatosis is a major source of morbidity and mortality in patients with advanced abdominal neoplasms. Intraperitoneal chemotherapy (IPC) is an area of intense interest given its efficacy in ovarian cancer. However, IPC suffers from poor drug penetration into peritoneal tumors. As such, extensive cytoreductive surgery is required prior to IPC. Here, we explore the utility of iRGD, a tumor-penetrating peptide, for improved tumor-specific penetration of intraperitoneal compounds and enhanced IPC in mice. Intraperitoneally administered iRGD significantly enhanced penetration of an attached fluorescein into disseminated peritoneal tumor nodules. The penetration was tumor-specific, circulation-independent, and mediated by the neuropilin-binding RXXK tissue-penetration peptide motif of iRGD. Q-iRGD, which fluoresces upon cleavage, including the one that leads to RXXK activation, specifically labeled peritoneal metastases displaying different growth patterns in mice. Importantly, iRGD enhanced intratumoral entry of intraperitoneally co-injected dextran to approximately 300% and doxorubicin to 250%. Intraperitoneal iRGD/doxorubicin combination therapy inhibited the growth of bulky peritoneal tumors and reduced systemic drug toxicity. iRGD delivered attached fluorescein and co-applied nanoparticles deep into fresh human peritoneal metastasis explants. These results indicate that intraperitoneal iRGD co-administration serves as a simple and effective strategy to facilitate tumor detection and improve the therapeutic index of IPC for peritoneal carcinomatosis.

Cisplatin (cDDP) is known to bind to the CXXC motif of proteins containing a ferrodoxin-like fold but little is known about its ability to interact with other Cu-binding proteins. MEK1/2 has recently been identified as a Cu-dependent enzyme that does not contain a CXXC motif. We found that cDDP bound to and inhibited the activity of recombinant MEK1 with an IC50 of 0.28 μM and MEK1/2 in whole cells with an IC50 of 37.4 μM. The inhibition of MEK1/2 was relieved by both Cu+1 and Cu+2 in a concentration-dependent manner. cDDP did not inhibit the upstream pathways responsible for activating MEK1/2, and did not cause an acute depletion of cellular Cu that could account for the reduction in MEK1/2 activity. cDDP was found to bind MEK1/2 in whole cells and the extent of binding was augmented by supplementary Cu and reduced by Cu chelation. Molecular modeling predicts 3 Cu and cDDP binding sites and quantum chemistry calculations indicate that cDDP would be expected to displace Cu from each of these sites. We conclude that, at clinically relevant concentrations, cDDP binds to and inhibits MEK1/2 and that both the binding and inhibitory activity are related to its interaction with Cu bound to MEK1/2. This may provide the basis for useful interactions of cDDP with other drugs that inhibit MAPK pathway signaling.

© The Royal Society of Chemistry. Mammalian cells have two influx Cu transporters that form trimers in membranes. CTR1 is the high affinity transporter that resides largely in the plasma membrane, and CTR2 is the low affinity transporter that is primarily associated with vesicular structures inside the cell. The major differences between CTR1 and CTR2 are that CTR1 contains a HIS/MET-rich domain N-terminal of the METS that participate in the first two stacked rings that form the pore, and a longer C-terminal tail that includes a Cu binding HIS-CYS-HIS (HCH) motif right at the end. It has been reported that CTR1 and CTR2 are physically associated with each other in the cell. We used the CRISPR-Cas9 technology to knock out either CTR1 or CTR2 in fully malignant HEK293T and OVCAR8 human ovarian cancer cells to investigate the interaction of CTR1 and CTR2. We report here that the level of CTR2 protein is markedly decreased in CTR1 knockout clones while the CTR2 transcript level remains unchanged. CTR2 was found to be highly ubiquitinated in the CTR1 knock out cells, and inhibition of the proteasome prevented the degradation of CTR2 when CTR1 was not present while inhibition of autophagy had no effect. Re-expression of CTR1 rescued CTR2 from degradation in the CTR1 knockout cells. We conclude that CTR1 is essential to maintain the stability of CTR2 and that in the absence of CTR1 CTR2 is degraded by the proteasome. This reinforces the concept that the functions of CTR1 and CTR2 are inter-dependent within the Cu homeostasis system.

Acquired resistance to carboplatin is a major obstacle to the cure of ovarian cancer, but its molecular underpinnings are still poorly understood and often inconsistent between in vitro modeling studies. Using sequential treatment cycles, multiple clones derived from a single ovarian cancer cell reached similar levels of resistance. The resistant clones showed significant transcriptional heterogeneity, with shared repression of cell cycle processes and induction of IFNα response signaling, and subsequent pharmacological inhibition of the JAK/STAT pathway led to a general increase in carboplatin sensitivity. Gene-expression based virtual synchronization of 26,772 single cells from 2 treatment steps and 4 resistant clones was used to evaluate the activity of Hallmark gene sets in proliferative (P) and quiescent (Q) phases. Two behaviors were associated with resistance: 1) broad repression in the P phase observed in all clones in early resistant steps and 2) prevalent induction in Q phase observed in the late treatment step of one clone. Furthermore, the induction of IFNα response in P phase or Wnt-signaling in Q phase were observed in distinct resistant clones. These observations suggest a model of resistance hysteresis, where functional alterations of the P and Q phase states affect the dynamics of the successive transitions between drug exposure and recovery, and prompts for a precise monitoring of single-cell states to develop more effective schedules for, or combination of, chemotherapy treatments.

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G0–G1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)3]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253)3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253)3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177–86. ©2018 AACR.

Purpose Intraperitoneal (IP) therapy improves survival compared to intravenous (IV) treatment for women with newly diagnosed, optimally cytoreduced, ovarian cancer. However, the role of IP therapy in recurrent disease is unknown. Preclinical data demonstrated IP administration of the proteasome inhibitor, bortezomib prior to IP carboplatin increased tumor platinum accumulation resulting in synergistic cytotoxicity. We conducted this phase I trial of IP bortezomib and carboplatin in women with recurrent disease. Methods Women with recurrent ovarian cancer were treated with escalating doses of IP bortezomib - in combination with IP carboplatin (AUC 4 or 5) every 21days for 6cycles. Pharmacokinetics of both agents were evaluated in cycle 1. Results Thirty-three women participated; 32 were evaluable for safety. Two patients experienced dose-limiting toxicity (DLT) at the first dose level (carboplatin AUC 5, bortezomib 0.5mg/m 2 ), prompting carboplatin reduction to AUC 4 for subsequent dose levels. With carboplatin dose fixed at AUC 4, bortezomib was escalated from 0.5 to 2.5mg/m 2 without DLT. Grade 3/4 related toxicities included abdominal pain, nausea, vomiting, and diarrhea which were infrequent. The overall response rate in patients with measurable disease (n=21) was 19% (1 complete, 3 partial). C max and AUC in peritoneal fluid and plasma increased linearly with dose, with a favorable exposure ratio of the peritoneal cavity relative to peripheral blood plasma. Conclusion IP administration of this novel combination was feasible and showed promising activity in this phase I trial of heavily pre-treated women with ovarian cancer. Further evaluation of this IP combination should be conducted.

INTRODUCTION: APTO-253 has captured attention because of its distinguishing mechanism of action as the only known a small molecule in clinical development to target a conserved structure in the promoter of the MYC oncogene and interrupt MYC gene expression. In preclinical studies of acute myeloid leukemia (AML) cell lines, APTO-253 was discovered to potently down-regulate MYC gene expression, reduce MYC mRNA and protein level, induce apoptosis, and deplete cells of the MYC oncoprotein. APTO-253 demonstrated broad killing of primary mononuclear cells isolated from the bone marrow of patients with AML, MDS or MPN and enhanced AML cell killing when combined with BET bromodomain inhibitors or FLT3 inhibitors (Kurtz, Blood 2017 126:1358). Because dysregulated MYC is considered a major oncogenic operator in AML and myelodysplasias and because APTO-253 has such a distinct mechanism to kill such cells, APTO-253 has been granted orphan drug designation for the treatment of AML by the US FDA and is currently in a Phase 1a/b clinical trial in patients with relapsed or refractory AML (R/R AML) or high-risk myelodysplasias (high-risk MDS). METHODS: APTO-253 is being evaluate in an ongoing clinical trial, entitled "A Phase I a/b Dose Escalation and Expansion, Multicenter, Open-label, Safety, Pharmacokinetic and Pharmacodynamic Study of APTO-253 in Patients with Relapsed or Refractory Acute Myelogenous Leukemia or High-Risk Myelodysplasia (NCT02267863)". The protocol provides for patients to be dosed once weekly on days 1, 8, 15, and 22 of each 28-day cycle with the intended dose levels of 20, 40, 66, 100, 140, 180 or 220 mg/m2. Eligible patients must be ≥18 years old, have a life expectancy of at least 2 months, and have acceptable hematologic, renal and liver functions and coagulation status parameters. Primary objectives are to determine the safety and tolerability of APTO-253, to determine the maximum tolerated dose (MTD) and the dose limiting toxicities (DLT), and to establish the recommended Phase 2 dose for patients with specific types of hematologic malignancies. Key secondary objectives are to seek evidence of antitumor activity by hematologic and bone marrow evaluations and to assess the impact of APTO-253 on the expression of pharmacodynamic biomarkers (including MYC and p21 gene expression). RESULTS: To date, R/R AML and high-risk MDS patients have been enrolled and treated at dose levels of 20, 40 and 66 mg/m2 APTO-253. As specified by the protocol, only one patient was required at each of the 20 and 40 mg/m2 doses. The AML patient on the lowest dose level completed the 28-day cycle and demonstrated approximately a 72% reduction in MYC expression levels in peripheral white blood cells during the cycle. The high-risk MDS patient who received 40 mg/m2 demonstrated a 79% reduction in MYC expression during the first cycle. To date (as of July 2019), no drug related TEAEs, SAEs or dose limiting toxicities have been reported. Two patients are receiving treatment with 66 mg/m2, and samples are being analyzed for a series of pharmacodynamic markers. CONCLUSIONS: APTO-253 is a potent molecule with a particularly interesting and novel mechanism of action that results in robust suppression of MYC expression. The Phase 1 a/b trial has successfully enrolled R/R-AML and HR-MDS patients into the first three cohorts at 20, 40 and 66 mg/m2. Clinical data to date suggest APTO-253 is generally well tolerated at the doses tested, and target engagement has been evidenced by the reduction in cellular MYC gene expression in whole blood samples from R/R AML and high-risk MDS patients. Recruitment and enrollment are continuing, and updated safety, PK and biomarker data will be presented at the meeting. Disclosures Howell: Aptose Biosciences, Inc: Consultancy, Research Funding. Zhang:Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

APTO-253 is a small molecule with a novel mechanism of action and potent antiproliferative activity against cell lines derived from a wide range of human malignancies. A Phase 1 study of APTO-253 was completed in patients with advanced solid tumors. It was well tolerated and produced evidence of antitumor activity in patients but did not cause myelosuppression even at the maximum tested dose. A phase 1a/b trial of APTO-253 in patients with relapsed/refractory acute leukemias (including AML) and high-risk MDS (NCT02267863) is currently underway. We previously reported that APTO-253 was converted intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. Both APTO-253 and Fe(253)3 stabilized DNA quadruplex (G4) structures in the Myc promotor region to reduce the expression of Myc (Figure 1). Stabilization of G4 structures has been reported to stall replication forks and produce DNA strand breaks. Treatment of cancer cells with APTO-253 produced time and concentration-dependent γH2AX foci formation and DNA double strand breaks that have to be repaired by homologous recombination. Loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 whose magnitude was similar to the effects of PARP inhibitors, olaparib. Thus, APTO-253 is a member of the limited repertoire of drugs which can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. The goal of this project was to identify synthetic lethal interactions in addition to BRCA1/2 deficiency that can guide combination drug studies. The normal function of the isocitrate dehydrogenase (IDH) enzymes is to catalyze the conversion of isocitrate to α-ketoglutarate (αKG) in the citric acid cycle. IDH1 mutations occur in more than 70% of low-grade gliomas, 20% of high-grade glioblastomas and with a frequency of about 10% in AML, cholangiocarcinomas, melanomas and chondrosarcomas. Additionally, mutations are also identified in IDH2 in about 4% of gliomas and 10% of AMLs. IDH1/2 alterations are heterozygous missense mutations that confer a neomorphic activity on the encoded enzyme such that they covert αKG to (R)-2-hydroxyglutarate [(R)-2HG]. (R)-2HG is an oncometabolite with pleiotropic effects on cell biology including chromatin methylation and cellular differentiation. It was reported that IDH1/2 mutations induce a homologous recombination defect that renders tumor cells exquisitely sensitive to PARP inhibitors. We used 2 pairs of isogenic IDH1 wild type and mutant cell lines to test the hypothesis that cells carrying IDH1/2 mutations are hypersensitive to APTO-253. Induction of the expression of IDH1 R132H in THP-1 AML cells increased the 2-HG concentration ~150-fold whereas induction of wild-type IDH1 expression increased it only 2-fold (doi: 10.1038/nm.3788). THP-1 cells expressing the mutant IDH1 R132H were hypersensitive the treatment of APTO-253 compared to cells expressing the wild-type IDH1 (Figure 2). Comparison of wild-type and isogenic IDH1 R132H HCT116 cells disclosed a ~100-fold higher level of (R)-2-HG in the mutant cells (doi: 10.1126/scitranslmed.aal2463), and an increase in sensitivity to the cytotoxic effect of APTO-253 (Figure 3). These results are consistent with the observation that both APTO-253 and increased levels of 2-HG cause DNA damage that depends on homologous recombination for repair and that this favors a synthetic lethal interaction. We surmise that the ability of APTO-253 to markedly reduce the expression of Myc further contributes to the favorable interaction. This data further supports the conclusion that defects in homologous DNA repair can enhance the potency of APTO-253 and suggest the use of this non-myelosuppressive drug in both IDH1/2 mutant AML and solid tumors. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Howell:Aptose Biosciences, Inc: Consultancy, Research Funding.

INTRODUCTION: CG-806 is a first-in-class, oral, non-covalent, and potent inhibitor of the wild type and all known mutant forms of Bruton's tyrosine kinase (BTK, including the C481S mutation), as well as all forms of the FLT3 receptor tyrosine kinase and multiple oncogenic signaling pathways, but with a selectivity that spares normal cells by avoiding off-target inhibition of kinases known to produce toxicities. Such a kinase inhibitory profile is key to the treatment of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and certain B-cell malignancies, which are driven by dysregulated BTK, can become resistant to covalent BTK inhibitors (ibrutinib and others) by BTK C481S mutation, and may become refractory to various therapies by other compensatory kinase signaling pathways such as SYK, SRC, STATs, ERK, AKT, NFĸB and MYC. In cell lines and primary samples from CLL and B-cell ALL patients, CG-806 potently suppressed the driver and compensatory pathways (inhibited phosphorylation of BTK, FLT3, PDGFRα, SYK, SRC, ERK, STAT5 and AKT, decreased MYC levels), induced cleavage of caspase 3, potently killed cells insensitive to ibrutinib or venetoclax, and caused enhanced cell killing in combination with venetoclax. Consequently, CG-806 is currently being evaluated in a Phase I a/b trial in patients with CLL/SLL or non-Hodgkin's lymphomas (NHLs). METHODS: A Phase 1 a/b trial of CG-806 in patients with CLL/SLL or non-Hodgkin's lymphomas (NCT03893682) is underway to evaluate the safety, tolerability and potential effectiveness of CG-806 for the treatment of patients for which either the standard treatment has failed, is no longer effective, or can no longer be administered safely. The starting dose is 150 mg PO BID and the dose will be escalated in 150 mg increments with only 1 patient at the two lowest dose levels and a minimum of 3 at the higher dose levels. Once the maximum tolerated dose or recommended dose is reached, up to 100 patients will be enrolled in the expansion cohort at the recommended dose. Primary objectives of the trial are to determine the safety and tolerability of CG-806 at the dose given orally every 12 hours that maintains a biologically active plasma concentration over a period of 28 days and to establish the recommended Phase 2 dose (RP2D) of CG-806 for future clinical trials in patients with advanced CLL/SLL or NHL. Key secondary objectives are to observe patients for any evidence antitumor activity using FDG PET-CT imaging evaluations and to measure pharmacodynamic biomarkers of drug effect including phospho-BTK levels and selected mRNA levels in peripheral white blood cells, as well as CCL3 and CCL4 circulating levels. RESULTS: CG-806 oral capsules have been administered at a starting dose of 150 mg BID on a 28-day cycle to the first CLL patient who previously failed ibrutinib, venetoclax, rituximab, idelalisib and other medications. To date (as of July 2019), in this first patient who has received more than 40 doses of CG-806 150mg oral capsules, no dose limiting toxicities have been observed, there are no impediments to continuing dosing on study, and the tolerability is consistent with preclinical tolerability findings in rodents and dogs when CG-806 was delivered as an oral gavage. Safety and tolerance vigilance, as well as pharmacokinetic (PK) and pharmacodynamic analyses, will continue into subsequent cycles and dose levels. CONCLUSIONS: CG-806 is an oral, non-covalent pan-BTK/pan-FLT3 inhibitor designed to suppress multiple oncogenic driver and compensatory signaling pathways in order to directly and potently kill B-cell cancer cells including those insensitive to the ibrutinib covalent BTK inhibitor and the venetoclax Bcl-2 inhibitor. Recruitment and enrollment of patients with R/R CLL/SLL and non-Hodgkin's lymphomas are continuing in a Phase 1a/b dose escalation/expansion trial, and updated safety, PK, and available biomarker data will be presented at the meeting. Disclosures Howell: Aptose Biosciences, Inc: Consultancy, Research Funding. Zhang:Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Despite the promise of ribonucleic acid interference therapeutics, the delivery of oligonucleotides selectively to diseased tissues in the body, and specifically to the cellular location in the tissues needed to provide optimal therapeutic outcome, remains a significant challenge. Here, key material properties and biological mechanisms for delivery of short interfering RNAs (siRNAs) to effectively silence target-specific cells in vivo are identified. Using porous silicon nanoparticles as the siRNA host, tumor-targeting peptides for selective tissue homing, and fusogenic lipid coatings to induce fusion with the plasma membrane, it is shown that the uptake mechanism can be engineered to be independent of common receptor-mediated endocytosis pathways. Two examples of the potential broad clinical applicability of this concept in a mouse xenograft model of ovarian cancer peritoneal carcinomatosis are provided: silencing the Rev3l subunit of polymerase Pol ζ to impair DNA repair in combination with cisplatin; and reprogramming tumor-associated macrophages into a proinflammatory state.

APTO-253 is a clinical stage small molecule that acts in part by down-regulation of MYC at mRNA and protein levels. MYC onco-protein dysregulation, which leads to increased proliferation and survival, is implicated in the etiology of numerous cancer subtypes. In the current study, we explored sensitivity of B-cell cancer cells to APTO-253 and sought to understand how changes in MYC can yield resistance to APTO-253. APTO-253 showed potency against B-cell cancer cell lines (IC50 30-600 nM) and bone marrow samples from patients. Sensitivity of cell lines correlated with MYC over-expression, amplification or translocation. APTO-253 resistant Raji cells (Raji253R) are characterized by upregulation of the ABCG2 transporter. Treatment with ABCG2 inhibitors only partially reversed the resistant phenotype suggesting additional contributing mechanisms. RNA-seq analysis revealed that while levels of MYC mRNA were unchanged in Raji253R, a truncation of exon 2 was present. This internal deletion occurred at the DNA level, not by alternative splicing, and resulted in a truncated MYC protein (45 kDa) lacking the conserved core sequence of MYC box III (MBIII). At early stages in the evolution of resistance residual full length MYC mRNA and protein was detectable; however, this expression was eventually lost in Raji253R cells as selection continued. Deletion of MBIII has been implicated in MYC protein stability, in vitro and in vivo transformation, gene repression, and MYC dependent apoptosis. Cycloheximide treatment of Raji and Raji253R cells demonstrated a modest increase in stability of truncated over full length MYC protein. In addition, Raji253R cells exhibited hypersensitivity to etoposide. The internal truncation is flanked by sites of microhomology suggesting a method for deletion. We hypothesize that DNA damage in response to APTO-253 treatment led to error prone repair by microhomology end joining and resulted in a MYC internal deletion. Interestingly, transcription from the canonical P1/P2 promoter, as seen in WT Raji cells, was systematically lost and transcription initiation shifted to a downstream promoter, P3. APTO-253 inhibits MYC expression via binding and stabilizing the G-quadruplex (G4) sequence in the P1/P2 promoter therefore switching of transcriptional initiation to P3 should relieve APTO-253 mediated repression. Indeed, APTO-253 treatment of Raji253R cells did not repress MYC expression, even at concentrations 50-fold higher than required for MYC repression in Raji WT cells. Presence of the active intra-cellular form of APTO-253, Fe(253)3, was confirmed in Raji253R cells. In summary, MYC driven Raji cells become resistant to APTO-253 by acquisition of a more stable MYC protein and by utilization of an alternate promoter not inhibited by G4 binding and stabilization, thereby confirming the essential role of MYC repression in the mechanism of action of APTO-253. Citation Format: Andrea Local, Cheng-Yu Tsai, Hongying Zhang, Susan Sheng, Khalid Benbatoul, Stephen B. Howell, William G. Rice. Resistance to APTO-253 caused by internal deletion and alternate promoter usage of the MYC gene in malignant B cells [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 2096.

CG-806 is a pan-FLT3 / pan-BTK inhibitor that is more potent (IC50 = 0.08 µM, n=265, p < 0.001) than other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib, crenolanib and gilteritinib against acute myeloid leukemia (AML) primary patient samples containing wild-type or mutated FLT3. CG-806 has significant activity against AML cell lines with or without FLT3 internal tandem duplication (ITD) / tyrosine kinase domain (TKD) mutations and in mouse AML xenograft models. Oral CG-806 has a desirable safety profile in the pre-IND studies of rodent and dog 28-day GLP toxicology, rodent respiratory and central nervous system safety, and the bacterial reverse mutation assay. The current study explored the relationship between genetic abnormalities in bone marrow and peripheral mononuclear cells isolated from AML patients and sensitivity to CG-806 using an ex vivo cytotoxicity assay. To correlate CG-806 sensitivity with clinical status, gene abnormalities and expression levels, whole exome sequencing (n=118) and RNA sequencing (n=111) were performed. CG-806 was equally potent against cells from patients in the adverse, intermediate and favorable risk groups (2017 ELN risk stratification), and cells from patients with relapsed or transformed AML (WHO classification) were as sensitive as those from patients with de novo AML. CG-806 had equivalent potency in cases of TP53 WT and TP53 mutations, whereas cases with TP53 mutations were resistant (FDR-corrected p Citation Format: Hongying Zhang, Andrea Local, Jeffrey W. Tyner, Stephen E. Kurtz, Beth Wilmot, Shannon Mcweeney, Brian J. Druker, Stephen B. Howell, William G. Rice. CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients [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 1323.