Race Oncology : Compelling Zantrene Preclinical Kidney Cancer Data

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ASX Announcement

Compelling Preclinical Kidney Cancer Results for Zantrene

• Zantrene found to kill a range of kidney cancer cells both on its own and in combination with existing cancer treatments
• When used in combination with Zantrene the kidney cancer drugs lenvatinib, cabozantinib and pazopanib showed greatly improved cell killing (synergy)
• These results support advancing Zantrene in human kidney cancer trials.

10 March 2022 - Race Oncology Limited ("Race") is pleased to share final results from the clear cell renal cell carcinoma (a dangerous form of kidney cancer) preclinical program led by eminent cancer researcher, Associate Professor Nikki Verrills of The University of Newcastle and Hunter Medical Research Institute (ASX announcement: 25 March 2021).

This research found that Zantrene on its own and in combination with known kidney cancer drugs can kill kidney cancer cells at clinically relevant concentrations. These results support advancing Zantrene into the clinic as a possible new treatment option for advanced kidney cancer patients.

Figure 1. Human clear cell renal cell carcinoma (kidney cancer). Image courtesy of Wikipedia.

Chief Scientific Officer, Dr Daniel Tillett said: "The results from Prof Verrills laboratory are highly encouraging and supportive of our clinical plans for Zantrene in kidney cancer. Advanced kidney cancer has a large unmet need for improved treatment options and Zantrene in combination with existing treatments may offer new hope for patients with this devastating disease."

Chief Executive Officer, Mr Phillip Lynch said, "We are again pleased to note Zantrene's effectiveness both in isolation and in combination with other known kidney cancer treatments. This result encourages clinical translation, and we look forward to determining an optimal approach for progressing clinical study."

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 Background

Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, comprising over 70% of renal tumours (Figure 2). While a relatively rare cancer, accounting for approximately 2% of global cancer diagnoses and deaths, it has more than doubled in incidence over the past half-century, and today is the ninth most common cancer in the developed world1.

Figure 2. Kidney cancer types and relative incidence. Image courtesy of Wikipedia.

Advanced/metastatic clear cell renal cell carcinoma occurs in 25-30% of people before diagnosis. The clinical signs of ccRCC are often mild or non-existent until the disease has spread throughout the body (metastasis)2. The most common organs for ccRCC to metastasize to lymph nodes, lungs, bones, liver and brain3. Late diagnosis remains a major challenge in the effective treatment of ccRCC.

Treatment of Clear Cell Renal Cell Carcinoma

Advanced ccRCC has a poor prognosis compared to many other cancers. While there have been major improvements in kidney cancer treatment in recent years, including the recent approval of immune therapies, the five-year survival rate for advanced ccRCC is still as low as 12%4. New treatments and drug combinations remain urgently needed to address what is often a devastating disease.

Importance of FTO in Clear Cell Renal Carcinoma

A recent preclinical study identified a synthetically lethal interaction between the Von Hippel‐Lindau (VHL) tumour suppressor protein and the m6A RNA demethylase Fatso/Fat Mass and Obesity Protein (FTO), in ccRCC5. Synthetic lethality occurs when the loss of either one of a pair of genes or proteins has little or no effect on the survival of the cell, but the loss of both proteins (or their activity) at the same time is lethal.

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VHL is inactivated in the majority of ccRCC (~90%)6, suggesting that the loss of FTO activity could prove lethal to cells lacking a functional FTO protein. Xiao et al found that FTO expression is increased in VHL-deficient ccRCC tumours, and genetic inactivation of FTO reduced the growth and survival of VHL-deficient cells5.

Zantrene has been recently identified as a potent inhibitor of FTO7 so may prove efficacious in the treatment of ccRCC with inactive VHL genes. This hypothesis was tested using Zantrene on an isogenic VHL mutant and wildtype ccRCC cell line. The potential for synergies with Zantrene and existing kidney cancer treatments was also explored.

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

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Study Highlights

1. Zantrene kills clear cell renal cell carcinoma cells

The sensitivity of kidney cancer and normal kidney cell lines to Zantrene was tested as a single agent to determine the cytotoxicity IC50 (drug concentration required to kill 50% of cells). Zantrene cytotoxicity was measured using a resazurin assay combined with visual inspection of the cells at each dose level and the IC50 values calculated. Direct cytotoxicity IC50 values ranged from 242nM to 12,353nM in the kidney cancer cell lines tested (Table 1). With the exception of the ccRCC A-704 cells, which were highly resistant, all other lines showed IC50 values below 1.4µM with 7 of the 12 lines displaying IC50 values below 1µM, a concentration achievable in patients based on prior human trials.

Table 1. Cytotoxic IC50 values of Zantrene in human renal cell lines.

Cell Line	Renal Cell Type	Zantrene IC50 (nM)
HK-2	Non-tumourigenic cortex/proximal	1061
	tubule
HEK293	Tumourigenic embryonic kidney	219
ACHN	Metastatic (pleural effusion)	242
Caki-1	Adenocarcinoma (metastatic)	659
Caki-2	Adenocarcinoma	331
769-P	ccRCC	1028
786-O	ccRCC	1309
A-704	ccRCC	12353
KMRC-1	ccRCC	506
A-498	ccRCC	325
RCC4 EV (VHL mutant)	ccRCC	907
RCC4 VHL (VHL widetype)	ccRCC	1170

Blue: non-cancer cell lines; Black: kidney cancer cell lines.

To determine if VHL status (i.e. wildtype or mutant/deleted) was associated with increased sensitivity to Zantrene, the isogenic cell lines RCC4 EV, which has a mutant VHL gene and the RCC4 VHL cell line which has been transduced with the wildtype VHL gene to rescue the VHL loss, showed that the VHL mutant cell line was more sensitive (1.3x) to Zantrene than the VHL rescue line (Figure 3).

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. Association between Zantrene sensitivity and VHL status. Direct comparison of Zantrene sensitivity in the RCC4 isogenic cell line pair. Cells were treated for 72h with indicated drug doses and cell viability determined. Mean +/- SEM, n=3.

Race Oncology Ltd ABN 61 149 318 749

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

www.raceoncology.com