On March 10, 2022 Race Oncology Limited ("Race") reported that 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) (Press release, Race Oncology, MAR 10, 2022, View Source [SID1234610002]).
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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 Registered office: L36, 1 Macquarie Place, Sydney NSW 2000 www.raceoncology.com 2 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 m6 A 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. Race Oncology Ltd ABN 61 149 318 749 Registered office: L36, 1 Macquarie Place, Sydney NSW 2000 www.raceoncology.com 3 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.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.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).2. Zantrene slows the growth of ccRCC cells Greater lethality between VHL loss and Zantrene was observed using long term clonogenic cell growth assays (cell colony formation), which better measures a drug’s effect on cancer cell growth rather than cell killing5.
The clonogenic assays were performed on the same panel of renal cell lines. A representative example of the effect of Zantrene on ccRCC cell colony formation is shown in Figure 4. Figure 4. Clonogenic cell growth assay of Zantrene in RCC4 EV cells. RCC4 EV cells were seeded in 6-well plates at 1000 cells/well and left to adhere overnight before treatment with the indicated concentrations of Zantrene for 96h. Wells had fresh media added and were left for an additional 96h to assess drug recovery.
Clonogenicity was assessed using crystal violet staining. Image of plate used for analysis (left). Colony area normalised to the untreated (UT) wells (right). Mean +/-SEM, n=3. All ccRCC cell lines were more sensitive to Zantrene (i.e. lower IC50 values) in the clonogenic cell growth assay (Table 2 & Figure 4). Similar sensitivity trends were observed as those seen in the cytotoxicity assay (Table 1). The A-704 cells remained the most resistant to Zantrene and the HEK293, ACHN, KMRC-1 and A-498 cell lines were the most sensitive. Interestingly, the Caki-1 ccRCC cells showed more than 10 times greater sensitive to Zantrene in the clonogenic assay than in the cytotoxicity assay (60nM verses 659nM) suggesting Zantrene may be a potent inhibitor of ccRCC growth at concentrations below the cytotoxic level. As seen in the cytotoxicity assays, the RCC-4 EV (VHL mutant) cells were significantly more sensitive (2.9x) to Zantrene than the wild-type RCC-4 VHL rescue cells (Table 2 & Figure 5). Race Oncology Ltd ABN 61 149 318 749 Registered office: L36, 1 Macquarie Place, Sydney NSW 2000 www.raceoncology.com 7 Figure 5. Association of Zantrene sensitivity and VHL status as assessed using clonogenicity assays. Colonies observed for the RCC4 EV and RCC4 VHL rescue cell lines.Cells were treated as described in Figure 4. NS, not significant, unpaired t-test. n=4, *p<0.05, paired t-test.
Table 2. Clonogenic IC50 of Zantrene in human renal cell lines. Cell Line Renal Cell Type Zantrene IC50 (nM) HK-2 Non-tumourigenic cortex/proximal tubule 217 HEK293 Tumourigenic embryonic kidney 46 ACHN Metastatic (pleural effusion) 53 Caki-1 Adenocarcinoma (metastatic) 60 Caki-2 Adenocarcinoma 116 769-P ccRCC 84 786-O ccRCC 229 A-704 ccRCC 750 KMRC-1 ccRCC 60 A-498 ccRCC 46 RCC4 EV (VHL mutant) ccRCC 167 RCC4 VHL (VHL widetype) ccRCC 483 Blue: non-cancer cell lines; Black: kidney cancer cell lines. Race Oncology Ltd ABN 61 149 318 749 Registered office: L36, 1 Macquarie Place, Sydney NSW 2000 www.raceoncology.com 8 3.
The FTO inhibitor Dac51 is less effective at killing ccRCC cells than Zantrene The cytotoxic sensitivity of five ccRCC cell lines to the structurally distinct FTO inhibitor, Dac519, was examined. A significantly higher concentration of Dac51 than Zantrene was required to kill all five ccRCC cell lines (Figure 6 & Table 3). Figure 6. Cytotoxicity of single agent Zantrene and DAC51 in clear cell renal cell carcinoma cell lines. 786-O, Caki-1, Caki-2, RCC4 EV and RCC4 VHL ccRCC cells were treated for 72h with the indicated drug concentrations of (A) Zantrene or (B) DAC51. Cell viability was determined using the resazurin metabolic assay and visual inspection. Cell viability is expressed as a percentage of untreated control cells. Mean +/-SEM, n=3. A number of interesting differences were noted between the two FTO inhibitor agents. The 786-O cell line was the most sensitive to Dac51, yet the least sensitive to Zantrene.
In contrast, the Caki-2 cells were the most sensitive to Zantrene, but the least sensitive to Dac51 (Table 3). Unlike Zantrene, Dac51 was more effective at killing the RCC4 VHL wildtype cell line than the corresponding mutant RCC4 EV cell line. In addition, the cytotoxic IC50 values for Dac51 ranged between 14x to 50x its reported IC50 values for FTO inhibition (0.4µM)9, suggesting that the modest Dac51 cytotoxic activity may reflect an offtarget effect.