NUS Presentation Title 2006

INTRODUCTION

l  Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by uncontrolled proliferation and a differentiation block of immature myeloid cells.

l  Differentiation therapies achieve remarkable success in acute promyelocytic leukemia (APL), a subtype of AML.

l  However, clinical benefits of differentiation therapies are negligible in non-APL AML, which accounts for the majority of AML cases.

l  Dihydroorotate dehydrogenase (DHODH) regulates the fourth step of the de novo pyrimidine synthesis pathway.

l  DHODH is a key therapeutic target for auto-immune diseases and cancer, particularly differentiation of AML1.

l  ASLAN003 is a novel, potent small molecule DHODH inhibi tor being developed in AML by ASLAN Pharmaceuticals.

ASLAN003 inhibits cell proliferation and induces cell differentiation of AML cell lines

Figure 2: The effects of ASLAN003, BRQ and addition of uridine on AML cells Figure 4: ASLAN003 shows excellent efficacy in orthotropic models of AML

1. Sykes DB, et al. Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia. Cell,167(1), 171-186 (2016).

CONCLUSION •  ASLAN003 is a novel, highly potent DHODH inhibitor that induces terminal differentiation,

inhibits cell growth and promotes cell death of AML blasts, including relapsed AML blasts. •  ASLAN003 prolongs survival and shows therapeutic effects in mice bearing different AML cell

lines and reduces leukemic burden in an AML PDX model. •  ASLAN003 is being evaluated in a Phase IIA clinical trial in AML patients (NCT03451084;

Poster 2676).

REFERENCE

Robust in vivo efficacy of ASLAN003

Table 1: ASLAN003 has potent effects on AML cells.

AML cell line IC50 (nM) for proliferation*

ED50 (nM) for differentiation^

THP-1 152 28 KG-1 382 56

MOLM-14 582 85

*AML cells incubated with DMSO or increasing concentrations of ASLAN003 for 48 hr. Cell proliferation was monitored using the CellTiter-Glo® Luminescent Cell Viability Assay (CTG assay, Promega). Fraction of cell viability relative to that of DMSO-treated cell was determined.

^AML cells with DMSO or increasing concentrations of ASLAN003 for 96 hr. Nitro Blue Tetrazolium (NBT) reduction assay in ASLAN003-treated vs DMSO-treated AML cell lines.

*^The experiments were performed in triplicates. GraphPad Prism 7.0 was used to calculate the IC50 and ED50 values by fitting the raw data points with sigmoidal nonlinear regression.

Figure 1: ASLAN003 induces morphologic changes of terminal differentiation and NBT positivity.

A ASLAN003 DMSO

KG-1

MOLM-14

THP-1

Wright-Giemsa staining

B ASLAN003 DMSO

NBT reduction assay

Cells were treated with DMSO or ASLAN003 100 nM for 96 hr, followed by Wright-Giemsa staining (A) and NBT reduction assay (B). Representative images were shown.

Comparison of ASLAN003 and BRQ in differentiation of AML and uridine rescue

(A) Comparison of the effects of ASLAN003 100 nM vs BRQ (brequinar) 100 nM to induce differentiation of MOLM-14 cells. The absolute increase of CD11b+ cells was calculated based on percentage of CD11b+ cells increased in treated samples compared to DMSO control. Representative FACS plots of BRQ- and DMSO-treated MOLM-14 cells were shown.

(B) Supplementation with uridine blocked ASLAN003-induced differentiation (B) and cell death (C) in MOLM-14 and THP-1 cells. Leukemic cells were incubated with DMSO, ASLAN003, or ASLAN003 + 50 µM uridine. ASLAN003 was used at 100 nM for MOLM-14 and 50 nM for THP-1. The percentages of CD11b+ cells (B) or viable cells (C) were illustrated and representative FACS plots were shown. The data was based on triplicates in different experiments (mean ± SD). *p < 0.05; n.s.: not significant.

l  FACS analysis showed ASLAN003 was approximately two-fold more potent than BRQ in inducing differentiation of AML cells.

l  Uridine could abrogate ASLAN003-induced cell differentiation as well as cell cytotoxicity, implying on-target specificity of ASLAN003.

ASLAN003 modulates expression of myeloid lineage transcription factor

Table 2: Clinical characteristics of AML and MDS patients and their responses to ASLAN003 in ex vivo assay.

ASLAN003 induces differentiation in primary AML blasts

Figure 3: ASLAN003 induces differentiation through induction of Runx1, Pu.1, Gif1 and repression of HoxA9, Gata1.

MOLM-14 cells were treated with DMSO or ASLAN003 at concentration of 50 nM and 100 nM for 96 hr. RNA was extracted, followed by cDNA synthesis and qRT-PCR analysis to determine expression of selected myeloid transcription factors. The expression of each gene was normalized with respective GAPDH expression in each sample, and DMSO-treated samples were set as baseline (n = 3, mean ± SD). *p < 0.05 ; **p < 0.01; n.s.: not significant.

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n.s.

Response Group

Clinical Characteristics Diagnosis Karyotype FLT3 NPM1

High sensitive (≥15%) AML-M1 t(9;22) WT WT AML-M5 Normal WT WT

AML 43% AML-M2 +8 ITD Mutant MDS 50% AML with MDS t(8;21) NA NA

AML-M5 Normal NA NA AML-M4 -7 WT WT

MDS Normal NA NA MDS Normal NA NA MDS Complex NA NA

Moderate sensitive (≥5% and < 15%)

AML-M2 Normal TKD NA AML-M1 -9 NA NA AML-M4 Normal WT WT

AML 43% Relapsed AML +13 ITD NA MDS 50% AML-M4 Inv(16) TKD WT

AML-M4 Inv(16) ITD WT MDS Normal NA NA MDS -7 WT WT MDS -13, +8 NA NA

Resistant (< 5%) AML-M5a +8 TKD Mutant AML 14% AML-M1 +11 WT WT

l  The healthy control sample was resistant to ASLAN003.

•  The response of primary BM cells to ASLAN003 was classified into 3 categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥ 15%; moderate sensitive: ≥ 5%, but < 15%; resistant: < 5%, by FACS analysis.

•  MDS: myelodysplastic syndromes; FLT3: fms-like tyrosine kinase; ITD: internal tandem duplication; TKD: tyrosine kinase domain; WT: wild type; NA: not available; NPM: nucleophosmin-1

A

0 5 1 0 1 5 2 0 2 5 3 00

2 0

4 0

6 0

8 0

1 0 0

D a y s p o s t in o c u la tio n

Pe

rce

nt

su

rviv

al

V e h ic le C o n t r o l

A S L A N 0 0 3

Log-rank p = 0.031

(n = 12) (n = 11)

MOLM-14 xenograft

Treatment

0 5 1 0 1 5 2 0 2 5 3 00

2 0

4 0

6 0

8 0

1 0 0

D a y s p o s t in o c u la tio n

Pe

rce

nt

su

rviv

al

V e h ic le C o n t r o l

A S L A N 0 0 3

Log-rank p < 0.0001

(n = 15) (n = 15)

THP-1 xenograft

Treatment

(A) NSG mice were transplanted with MOLM-14 or THP-1 AML cells via tail vein injection. Mice were administrated with either ASLAN003 50 mg/kg or same volume of vehicle by daily oral gavage. Treatment was started 3 days after inoculation of leukemic cells. Kaplan-Meier survival curves of mice treated with either ASLAN003 or vehicle control for these two xenograft models were plotted. The number of mice in each group and Log-rank p values were indicated.

(B, C) The leukemic burden in mouse bone marrow (BM), peripheral blood (PB), liver and spleen were compared for the ASLAN003-treated and vehicle-treated control groups from 3 mice each group for MOLM-14 xenograft models and 4 mice each group for THP-1 xenograft models. Human CD45+ cells determined by FACS analysis as a surrogate marker for leukemic burden. FACS analysis was performed to assess the percentage of human specific CD11b+, CD14+ cells in BM samples harvested from these mice. P values were shown in each graph. p < 0.05 is statistically significant.

l  Significantly prolonged survival was observed in ASLAN003-treated groups when compared to vehicle control group in MOLM-14 (p = 0.031) and THP-1 (p < 0.001) orthotopic xenograft models.

l  ASLAN003 substantially reduced leukemic burden (human CD45+ cells) in BM, peripheral blood, liver and spleen, accompanied with significantly increased differentiation of AML cells (CD11b and CD14 positive cells) in BM of treated mice in both models.

Figure 5: ASLAN003 significantly decreased engraftment of primary AML cells in AML-14 PDX line

AML-14 Clinical information: FAB subtype: AML-M4; Karyotype: Normal; Gene mutation: FLT3, NPM1 status not available

A total of 3 million BM cells of AML-14 was injected into each NSG mouse via tail vein injection. The recipient mice were subjected to sub-lethal irradiation (2 Gy) 48 hours prior to inoculation. Mice were treated with ASLAN003 50 mg/kg, oral gavage daily, or same volume of vehicle as control. Treatment was started 5 days post inoculation. There were 5 mice in control group and 4 mice in ASLAN003-treatment group.

l  At day 77 post treatment, all PDX mice were alive in both control and ASLAN003 group. The leukemic burden was significantly lower in ASLAN003-treated PDXs than in vehicle-treated PDXs.

DISCLOSURES

Seet: ASLAN Pharmaceuticals: Employment, Equity Ownership. Ooi: ASLAN Pharmaceuticals: Employment, Equity Ownership. Lindmark: ASLAN Pharmaceuticals: Employment, Equity Ownership. McHale: ASLAN Pharmaceuticals: Employment, Equity Ownership. Chng: ASLAN Pharmaceuticals: Research Funding. The other authors declare no conflicts of interest.

RESULTS

B

Control ASLAN003

MOLM-14 xenograft

Control ASLAN003

Posi

tive

cells

(%)

hCD45+ in BM

p = 0.011

hCD45+ in PB

p = 0.004

hCD45+ in Spleen

Control ASLAN003

p = 0.033

p = 0.010

hCD45+ in Liver

hCD11b+ in BM hCD14+ in BM

p < 0.001 p = 0.003

Posi

tive

cells

(%)

Control ASLAN003 Control ASLAN003

Control ASLAN003

Posi

tive

cells

(%)

Posi

tive

cells

(%)

hCD45+ in BM hCD45+ in PB

p = 0.014 p = 0.013

hCD45+ in Spleen

p = 0.026

hCD45+ in Liver

p = 0.014

hCD11b+ in BM

p = 0.002 p = 0.013

Posi

tive

cells

(%)

hCD14+ in BM

THP-1 xenograft C

Control ASLAN003 Control ASLAN003

Control ASLAN003 Control ASLAN003

Control ASLAN003 Control ASLAN003

hCD45+ in BM

Posi

tive

cells

(%)

p = 0.0399

Control ASLAN003

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ASLAN003, a Novel and Potent Dihydroorotate Dehydrogenase (DHODH) Inhibitor, Induces Differentiation of Acute Myeloid Leukemia Jianbiao Zhou1.2, Jessie Yiying Quah1, Jing-Yuan Chooi2, Sabrina Hui-Min Toh1, Yvonne Ng1, Baohong Lin3, Motomi Osato1, Qihui Seet4, A.G. Lisa Ooi4, Bertil Lindmark4, Mark McHale4, and Wee-Joo Chng, M.D1,2,3

1Cancer Science Institute of Singapore, 2Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; email: csizjb@nus.edu.sg, 3Department of Hematology-Oncology, National University Cancer Institute, NUHS, Singapore. 4ASLAN Pharmaceuticals, Singapore

Publication Number: 4047