Race Oncology : Zantrene highly effective in EMD AML Mouse Model

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ASX Announcement

Zantrene Highly Effective in a Mouse Model of

Extramedullary AML

• Zantrene in combination with decitabine was found to target extramedullary tumours as well as in the bone marrow and spleen using an AML mouse model.
• Zantrene alone found to kill a genetically diverse range of AML cells at low drug concentrations and slow the growth of AML tumours in mice.
• Zantrene in combination with decitabine showed significantly greater cell killing across a diverse panel of AML cell lines than either drug on its own (true synergy).
• These results provide further robust in vitro and in vivo preclinical support for Race's announced extramedullary AML Phase 1/2 clinical trial.

17 March 2022 - Race Oncology Limited ("Race") is pleased to share interim results from the extramedullary acute myeloid leukaemia (EMD AML) preclinical program led by eminent cancer researcher, Associate Professor Nikki Verrills of The University of Newcastle and Hunter Medical Research Institute (ASX announcement: 30 March 2021).

This research found that low dose Zantrene in combination with decitabine can kill AML tumours in a mouse model of extramedullary AML and builds on the results of earlier AML clinical trials. Race is rapidly advancing Zantrene into the clinic as a possible new treatment option for patients with EMD AML.

Figure 1. Human AML cells (purple) from a bone marrow aspirate.

Chief Scientific Officer, Dr Daniel Tillett said, "The results from Prof Verrills laboratory are highly supportive of our upcoming EMD AML Phase 1/2 trial for Zantrene. This work further builds on the 2020 Phase 2 trial of Prof Arnon Nagler, who identified Zantrene as showing encouraging efficacy in EMD AML. The optimised drug combination and schedule identified in this preclinical mouse study will be rapidly translated to the clinic via our EMD AML trial."

Chief Executive Officer, Mr Phillip Lynch said, "These results provide support for our well advanced EMD AML clinical trial and provides important guidance for the study's design and treatment protocol."

Race Oncology Ltd ABN 61 149 318 749

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Study Background

Extramedullary AML

Extramedullary AML occurs when the leukaemia spreads from the bone marrow and forms solid tumours in tissues such as the skin, breast, kidney, brain, or other organs1. A 2020 prospective positron imaging trial (18FDG PET) identified that up to 22% of AML patients have the extramedullary form2. Extramedullary AML patients have no clinically approved treatments and only limited experimental treatment options3. Many clinical trials exclude patients with this difficult to treat form of AML.

Zantrene showed positive efficacy in extramedullary AML

In a 2020 Phase 2 clinical trial conducted at Chaim Sheba Medical Centre, Tel Aviv in relapsed and refractory (R/R) AML patients, Zantrene was observed to have clear efficacy in patients with extramedullary AML4. In this trial of 10 heavily pre-treated AML patients, all four patients having the EMD AML subtype showed a clinical response to a single cycle of Zantrene as a single agent4. An example 18FDG PET image of an EMD AML patient from this trial before and after Zantrene is shown in Figure 2.

Figure 2. Extramedullary AML 18FDG PET imaging before (left) with AML cancer cells appearing as dark /bright spots in breast tissue and after (right) a single cycle of treatment with Zantrene alone.

Synergy between FTO inhibition and decitabine

Su et al. (2018) observed synergy between FTO inhibition and decitabine against AML cells5. Decitabine is often used for the treatment of AML or myelodysplastic syndrome (MDS) in patients unfit for high intensity chemotherapy and stem cell transplantation6. On the basis that Zantrene is a potent FTO inhibitor (IC50 142nM)7, it was hypothesised that Zantrene and decitabine would synergise to better kill AML cells. This hypothesis was tested both in vitro in AML cell cultures and in vivo in a mouse model of extramedullary AML.

Race Oncology Ltd ABN 61 149 318 749

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Registered office: L36, 1 Macquarie Place, Sydney NSW 2000

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Study Highlights

1. Zantrene kills AML cells more effectively than decitabine as a single agent

Cells were treated for 72 hours and cell viability determined using a resazurin metabolic assay and visual inspection (Figure 3A). The concentration of drug required to inhibit cell viability by 50% (IC50) or 25% (IC25) was determined (Table 1).

Table 1. Single agent cytotoxicity of Zantrene and decitabine in a diverse range of human AML cell lines.

Cell Line

Driver Mutations

Isolation

Zantrene

Decitabine

IC25 (nM)

IC50 (nM)

IC25 (nM)

IC50 (nM)

MV4-11

FLT3-ITD

Diagnosis

4

16

81

> 500

Kasumi-1

KIT N822K

2nd

87

563

112

> 500

TP53

Relapse

ASXL1 c.1934dupG

THP-1

NRAS G12D

Relapse

95

152

156

> 500

TP53 del

HL-60

NRAS Q61L

Diagnosis

16

65

191

> 500

MYC amp

TP53 del

CDKN2A pR8

NOX5

MOLM-13

FLT3-ITD (Het)

Relapse

26

42

23

53

OCI-AML3

NPM1, DNMT3A

Diagnosis

71

124

>500

>500

R882C(het) NRAS

Q61L

All AML cell lines were sensitive to Zantrene in the nanomolar range at drug concentrations of clinical relevance (Table 1). The MV4‐11 cells were the most sensitive to single agent Zantrene, with an IC50 of just 16nM, followed by the MOLM-13 and HL60 cells at 42nM and 65nM, respectively. The OCI-AML3 and THP-1 lines showed similar sensitivity, with IC50s of 124nM and 152nM, respectively. The Kasumi-1 cells were more resistant to Zantrene with an IC50 of 563nM.

In contrast, decitabine treatment alone, up to 500nM for 72 hours, did not reach the IC50 level even at the 500nM concentration in 5 out of the 6 cell lines tested (Figure 3B & Table 1). The MOLM-13 cells were the most sensitive to decitabine with an IC50 of 53nM, however, 100% cell death was not achieved for decitabine at any tested dose.

Race Oncology Ltd ABN 61 149 318 749

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Registered office: L36, 1 Macquarie Place, Sydney NSW 2000

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Figure 3. Single agent Zantrene and decitabine cytotoxicity assays in human AML cells. Cells were treated for 72h with different dose combinations of (A) Zantrene or (B) Decitabine. Dose-responsecurves show cell viability in response to indicated dose ranges. n ≥2.

The MV4-11and MOLM-13cell lines both contain the fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD)mutation, suggesting a possible connection between FLT3- ITD mutations and Zantrene sensitivity. These two cell lines, as well as the THP-1cells also have MLL rearrangements, suggesting a further possible association of Zantrene sensitivity and MLL rearrangement. The HL60, THP-1and Kasumi-1lines all have tumour protein p53 (TP53) mutations, suggesting an association with TP53 mutational status and Zantrene sensitivity. Importantly, these data suggest that TP53 mutation alone does not induce resistance to Zantrene. The three cell lines with neuroblastoma RAS oncogene (NRAS) mutations were all sensitive to Zantrene (THP-1, HL60 and OCI-AML3).

In addition to having a TP53 deletion, the Kasumi-1 cell line (least sensitive to Zantrene) expresses the tyrosine-proteinkinase KIT (KIT) carrying a mutation within the tyrosine kinase domain causing constitutive activation of KIT, an oncogenic driver of AML.

The in vitro cell line data shows that Zantrene is able to kill AML cells at low concentrations as a single agent irrespective of the cancer driver mutations that are common causes of AML in patients with the possible exception of KIT.

2. Zantrene and decitabine work together to better kill human AML cell lines

The viability of AML cells to combined treatment with Zantrene and decitabine for 72 hours was analysed for synergy using the methods of Bliss which indicated several synergistic dose combinations in all AML cell lines tested (Table 2). An example of the Bliss analysis is shown in Figure 4 for the THP-1 and MOLM-13 cell lines.

Race Oncology Ltd ABN 61 149 318 749

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Registered office: L36, 1 Macquarie Place, Sydney NSW 2000

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Figure 4. Bliss Synergy Analysis for human THP-1 and MOLM-13 AML cells. (A) 2D and 3D visualisation of predicted Bliss scores at each dose point, with red to green scale indicating areas of synergy to antagonism, and the average synergy score. The most synergistic 2x2 area is indicated with a white box. (B) Table of Bliss scores for each individual dose combination. Values >10 are considered synergistic (red); values below -10are considered antagonistic. Values between -10and 10 are additive.

Race Oncology Ltd ABN 61 149 318 749

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Registered office: L36, 1 Macquarie Place, Sydney NSW 2000

www.raceoncology.com