ALBERT

Description: APTO-253, a small molecule that mediates anticancer activity through induction of the Krüppel-like factor 4 (KLF4) tumor suppressor, is being developed clinically for the treatment of acute myelogenous leukemia (AML) and high risk myelodysplastic syndromes (MDS). APTO-253 was well tolerated in a Phase I study in patients with solid tumors using a dosing schedule of days 1, 2, 15, 16 of a 28 day cycle (2T-12B-2T-12B), but recent scientific observations guided APTO-253 toward AML and high risk MDS. Indeed, suppression of KLF4 was reported as a key driver in the leukemogenesis of AML and subsets of other hematologic diseases. The vast majority (~90%) of patients with AML aberrantly express the transcription factor CDX2 in human bone marrow stem and progenitor cells (HSPC) (Scholl et al., J Clin Invest. 2007, 117(4):1037-48). The CDX2 protein binds to CDX2 consensus sequences within the KLF4 promoter, thereby suppressing KLF4 expression in HSPC (Faber et al., J Clin Invest. 2013, 123(1):299-314). Based on these observations, the anticancer activity of APTO-253 was examined in AML and other hematological cancers. APTO-253 showed potent antiproliferative activity in vitro against a panel of blood cancer cell lines, with ηM IC50values in AML (6.9 - 305 ηM), acute lymphoblastic leukemia and chronic myeloid leukemia (39 – 250 ηM), non-Hodgkin’s lymphoma (11 – 190 ηM) and multiple myeloma (72 – 180 ηM). To explore in vivo efficacy, dose scheduling studies were initially conducted in the H226 xenograft model in mice. In the H226 model, APTO-253 showed improved antitumor activity when administered for two consecutive days followed by a five day break from dosing (2T-5B) each week, i.e. on days 1,2, 8,9, 15,16, 22,23, compared to the 2T-12B-2T-12B schedule. The 2T-5B schedule was used to evaluate antitumor activity of APTO-253 in several AML xenograft models in mice. In Kasumi-1 AML and KG-1 AML xenograft models, APTO-253 showed significant antitumor activity (p = 0.028 and p=0.0004, respectively) as a single agent when administered using the 2T-5B schedule each week for four weeks compared to control animals. Mice treated with APTO-253 had no overt toxicity based on clinical observations and body weight measurements. Mice bearing HL-60 AML xenograft tumors were treated with APTO-253 for one day or two consecutive days per week for three weeks, either as a single agent or combined with azacitidine, or with azacitidine alone twice per week (on days 1,4, 8, 11, 15 and 18). APTO-253 as a single agent inhibited growth of HL-60 tumors to approximately the same extent as azacitidine. Furthermore, both once weekly and twice weekly dosing of APTO-253 in combination with azacitidine resulted in significantly enhanced antitumor activity relative to either single agent alone (p = 0.0002 and p = 0.0006 for 1X and 2X weekly APTO-253 treatment, respectively, compared to control). Likewise, using a THP-1 AML xenograft model, APTO-253 administered as a single agent using the 2T-5B per week schedule showed significant efficacy, similar to that of azacitidine, while the combination of APTO-253 and azacitidine demonstrated greatly improved antitumor effects relative to either drug alone. APTO-253 was effective and well tolerated as a single agent or in combination with azacitidine in multiple AML xenograft models, plus APTO-253 does not cause bone marrow suppression in animal models or humans. Taken together, our results indicate that APTO-253 may serve as a targeted agent for single agent use and may provide enhanced efficacy to standard of care chemotherapeutics for AML and other hematological malignancies. Disclosures Rice: Lorus Therapeutics Inc.: Employment. Vellanki:Lorus Therapeutics Inc.: Employment. Lee:Lorus Therapeutics Inc.: Employment. Lightfoot:Lorus Therapeutics Inc.: Employment. Peralta:Lorus Therapeutics Inc.: Employment. Jamerlan:Lorus Therapeutics Inc.: Employment. Jin:Lorus Therapeutics Inc.: Employment. Lum:Lorus Therapeutics Inc.: Employment. Cheng:Lorus Therapeutics Inc.: Employment.

Description: APTO-253 is a novel anticancer small molecule currently in a multicenter open-label, Phase I dose escalation study in patients with relapsed or refractory hematologic malignancies. APTO-253 has potent cytotoxic activity against leukemia, lymphoma and myeloma cell lines IC50s of 6.9 - 305 nM. APTO-253 produces significant tumor growth inhibition in the KG-1, THP-1 and Kasumi-1 xenograft models of human acute myeloid leukemia (AML). The anticancer activity of APTO-253 is mediated through induction of Krüppel-like factor 4 (KLF4), a tumor suppressor that is epigenetically silenced in many solid tumors and hematologic cancers. KLF4 expression is often downregulated in AML due to repressive binding of CDX2 to the KLF4 promoter. Increased levels of CDX2 are found in the majority of patients with AML and ALL, as well as in 40% of MDS patients, whereas CDX2 is not expressed in normal hematopoietic cells. Treatment of cultured AML cells with APTO-253 reverses the KLF4 silencing, resulting in induction of p21 and cell death by apoptosis. KG-1 AML cells treated with APTO-253 showed time- and concentration-dependent induction of KLF4 and a concentration-dependent increase in p21 mRNA levels following induction of KLF4. APTO-253 treatment of KG-1 cells for 24 h induced a 14-fold increase in KLF4 mRNA and 16-fold increase in p21 mRNA over basal levels. Following washout of APTO-253, the level of KLF4 mRNA decayed to approximately 50% of maximum induction level over a 24 h period. The potential for APTO-253 as a therapeutic option in AML was further supported by safety and pharmacokinetics data from an earlier Phase I trial of APTO-253 in patients with advanced or metastatic solid tumors during which APTO-253 was administered at doses of 20 - 387 mg/m2 using a dosing schedule of days 1 and 2, and 15 and 16 of a 28 day cycle. APTO-253 showed a dose-dependent increase in Cmax and AUC, and as the dose was escalated from 80 to 176 mg/m2 the Cmax ranged from 1,800 - 4,960 nM on day 1 and 1,600 - 6,100 nM on day 2. These results suggest that exposure levels from these doses of APTO-253 should be sufficient for single agent activity in patients with AML and other hematologic malignancies. APTO-253 demonstrated a favorable safety profile when tested against 5 major cytochrome P450 enzymes (1A2, 2C19, 2C9, 2D6 and 3A4), against a panel of proteins and receptors, and in the hERG tail current density assay. Metabolic profiling of APTO-253 at 50 μM in human liver microsomes showed no glutathione or glucuronide conjugation and only a minor hydroxylated metabolite. Finally, 1 μM APTO-253 did not inhibit kinases in a safety panel (40 kinases) or in a broad oncology panel (98 kinases), demonstrating that APTO-253 activity is not driven by kinase inhibition. Taken together, our results demonstrate that APTO-253 has substantial potential for the treatment of AML and other hematologic malignancies and is of particular interest due to its ability to modulate the expression of the KLF4 master transcription factor that plays a central role in restraining the growth of leukemic cells. Disclosures Howell: Aptose Biosciences: Consultancy, Equity Ownership; Angstrom: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Abeoda: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; InhibRx: Equity Ownership.

Description: The Bromodomain and Extra-Terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are functional readers of acetylated lysine residues of histones, and have emerged as potential therapeutic targets in hematologic cancers and solid tumors characterized by dysregulated epigenetic processes. Targeted inhibition of BET proteins has proven to be an effective strategy for transcriptional downregulation of c-MYC, an oncogene that is frequently activated or overexpressed in leukemias, lymphomas, and multiple myeloma. Of the BET family members BRD4 is the most extensively studied for its role in cancer, furthermore C-MYC downregulation by BET inhibitors is attributed to inhibition of enhancer binding by BRD4 (Delmore et al., Cell. 2011. 146:904-17). BRD4 is a critical factor in AML disease maintenance (Zuber et al., Nature. 2011. 478:524-8), and its suppression is the dominant mechanism of BET inhibitor JQ1 activity in AML (Rathert et al., Nature. 2015. 525:543-7). Recent reports have shown that BRD4/BET inhibitors and kinase inhibitors act synergistically in a range of cancer types (Sun et al., Blood. 2015, 126:1565-74; Stratikopoulos et al., Cancer Cell. 2015, 27:837-51). Therefore, optimizing for this synergy by prospectively designing and developing multi-targeting BRD4-kinase inhibitors may prolong therapeutic efficacy and overcome tumor resistance of single-activity BET and oncogenic kinase inhibitors. Dual inhibitors of BET proteins and Janus kinase 2 (JAK2), initially developed by Moffitt Cancer Center (Reuther et al., ASH 2015 Poster, Abstract #2826), demonstrated an opportunity for novel, potent dual inhibitors of BRD4 and JAK for treatment of myeloproliferative neoplasms (MPNs) and other disorders driven by the constitutively active somatic mutation, JAK2-V617F. Although JAK inhibitors such as ruxolitinib show clinical benefits in MPNs, these molecules demonstrate limited ability to induce remissions and are associated with significant toxicities such as myelosuppression. Therefore, selective JAK2 inhibitors that also target BRD4 hold promise as treatments of hematologic malignancies with improved activity and less off-target toxicity. While the dual inhibitor candidates from Moffitt exhibit strong potency, they possess a sub-optimal profile for inhibition of the thiamine transporter and other properties. Herein, we report that next generation, novel dual inhibitors of BRD4 and JAK2 have been discovered by Aptose, in a collaboration with Laxai Avanti Life Sciences (LALS), without observed thiamine transporter inhibition but with favourable drug-like properties. In biochemical assays, the Aptose dual inhibitor compounds exhibit IC50 values ranging from 82-678 nM for BRD4 (BD1) and 0.7-35 nM for JAK2. Compounds exhibited selectivity of 2-7 fold for BRD4 compared to other BRD isoforms, and 3-152 fold selectivity for JAK2 over JAK1 or JAK3. Cellular IC50 values of the compounds in antiproliferative assays ranged from 6.6 - 118 nM in AML (MV4-11) and from 171-721 nM in myeloma (MM1.S) cell lines. Candidate structures show good solubility and metabolic stability in human, rat and mouse liver microsomes. Structure-activity relationship studies are ongoing to improve biochemical and antiproliferative activity and drug like properties. Collectively, we demonstrate that a rational design approach can be used to generate potent molecules with combined BRD4/JAK2 inhibitory activities for treatment of hematologic cancers. Disclosures No relevant conflicts of interest to declare.

Description: The c-Myc multifunctional transcription factor protein, a product on the c-myc proto-oncogene, contributes to the pathogenesis of many types of human cancers through mechanisms of proliferation, apoptosis, cell cycle progression and cellular senescence. c-Myc is frequently overexpressed in acute myeloid leukemia, yet strategies to effectively modulate c-Myc function do not exist. We evaluated inhibition of c-myc gene expression by APTO-253, a small molecule anticancer agent that is being developed clinically for the treatment of acute myelogenous (myeloid) leukemia (AML) and high risk myelodysplastic syndromes (MDS). We first confirmed that c-Myc mRNA level were significantly higher in AML cell lines as compared to peripheral blood mononuclear cells (PBMCs) isolated from healthy human donors. However, the c-Myc expression in AML cells was inhibited by APTO-253 in dose-dependent and time-dependent manners at both the mRNA and protein levels. Likewise, APTO-253 was found to induce AML cell apoptosis in dose-dependent and time-dependent manners as demonstrated by positive Annexin-V staining and increases in cleaved poly (ADP-ribose) polymerase (c-PARP). APTO-253 induced AML cells arrest at G1/G0 phase of cell cycle by increasing p21 expression and decreasing expression of cyclin D3 and cyclin-dependent kinases 4/6 (CDK4/6). For the p53 positive cell lines MV4-11 and EOL-1, p53 was also increased by APTO-253 at early time points (less than 6-hour treatment), suggesting that p53-dependent cell cycle arrest and apoptosis is mechanistically operative as a consequence of treatment with APTO-253. Importantly, we demonstrated that APTO-253 selectively targeted tumor cells but not normal healthy cells, with MV4-11 AML cells and normal PBMCs having IC50s of 0.25±0.03µM and more than 100µM, respectively. Our previous studies (56th ASH abstract #4813) showed that APTO-253 induces the Krüppel-like Factor 4 (KLF4) transcription factor and was effective and well tolerated as a single agent in multiple AML xenograft models without causing bone marrow suppression. Taken together, our results suggested that APTO-253 may serve as an effective and safe agent for AML chemotherapy, and that APTO-253 mechanistically inhibits c-Myc expression in AML cells and subsequently induces cell cycle arrest and apoptosis. Disclosures No relevant conflicts of interest to declare.