Imagine a lock that changes its combination every time you try to open it. This is the common challenge faced by scientists and medical professionals whilst combating cancer. The deadly disease adapts to various therapies, surviving intense radiation, chemotherapy, targeted drugs, and more. This extraordinary ability of cancer cells aids them in developing resistance to various therapeutic avenues, and is especially detrimental in aggressive forms of cancer. Glioblastoma multiforme (GBM) is a prime example of an aggressive brain cancer, notorious for its resilience against treatment. At the Indian Institute of Technology Gandhinagar, researchers from the Cancer Chemical Biology Lab have been exploring the potential of the Tousled-like kinase 1 (TLK1), an enzyme known to repair damaged DNA, as a target for various types of cancers, including GBM.

TLK1 is part of the molecular machinery that facilitates the repair of DNA damage caused by carcinogens or cancer therapies, thereby promoting tumor cell survival. As a result, it has emerged as a potential therapeutic target to enhance the effectiveness of DNA-damaging treatments. Interestingly, research has indicated that in cancer cells, TLK1 seems to work overtime, patching up the DNA damage caused by anti-cancer drugs, boosting the survival of the diseased cells. The protein was shown to be an effective target of anti-cancer molecules in breast and prostate cancer, as indicated by previous studies conducted by the group individually and in collaboration with the De Benedetti Lab in LSU Health Shreveport (USA). “Conventional chemotherapy for GBM uses temozolomide (TMZ) to damage cancer cells’ DNA,” explained Dr Bhanu Priya, a PhD graduate from the Cancer Chemical Biology Lab and the first author of the study. Unfortunately, over time, some glioblastoma cells find ways to fix that damage or dodge it altogether. These “resistant” cells survive, multiply, and eventually render the drug ineffective. “Temozolomide resistance is one of the biggest challenges in glioblastoma therapy,” said Dr Sivapriya Kirubakaran, Professor at the Department of Chemistry and the Principal Investigator of the Cancer Chemical Biology Lab. “Once resistance sets in, the treatment options become extremely limited.”

“We sought to understand the mechanisms behind this resilience to create better treatment strategies that circumvent this challenge,” added Dr Priya, who is currently working as a Business Development Specialist at Topia Life Sciences, a pharmaceutical company in Ahmedabad, Gujarat. Their findings, recently published in Scientific Reports, also highlighted the potential of an in-house developed small molecule inhibitor, J54, to re-sensitise resistant tumour cells to treatment. To understand these molecular mechanisms, the IITGN team created TMZ-resistant cancer cells by exposing them to increasing doses of the drug over six months. During this prolonged exposure, where tumours are subjected to repeated chemotherapy, most of the cancer cells are killed by the drug, but a few manage to survive. These surviving cells adapt over time, developing ways to protect themselves, repair damage, or avoid being destroyed, replicating the resistant glioblastoma condition within a lab setting. As a result, they are able to withstand further TMZ treatments, and also exhibit noticeable differences in shape, behaviour, and molecular activity.

Detailed molecular analysis revealed high levels of TLK1. “We hypothesised that this DNA repair protein enables resistant cancer cells to repair the very damage TMZ is supposed to cause, helping the cells survive genetic damage and thrive,” explained Dr Kirubakarn. The team tested this hypothesis by treating resistant glioblastoma cells with J54, a TLK1 inhibitor developed in-house by former PhD scholars of the Cancer Chemical Biology Lab. The addition of J54 crippled the resistant cell’s ability to repair its DNA, leading to damaged genome integrity and triggering cell death. The cells which were unaffected by TMZ suddenly became vulnerable to the J54 again and significantly lost their ability to move and invade surrounding cells, a critical trait in glioblastoma’s uncontrollable and deadly spread.

The potent inhibitory effect of J54 on the protein is aided by the molecule’s chemical structure. When designed, J54 was based on a class of compounds (phenothiazines) which have proven use in the treatment of psychosis. Thus, it can cross the blood-brain barrier successfully, bypassing the shield that prevents many drugs from reaching the brain. Yet crossing this barrier is only the first step. To be truly effective, a therapeutic molecule must also intervene in the complex molecular machinery that fuels cancer survival.

Shedding more light on the complexity of cancer pathways, Dr Priya remarked, “The molecular players associated with cancer work in tandem. Thus, it is sometimes necessary to target downstream partners of a critical enzyme to ensure the success of your therapeutic strategy.” In line with this, the team performed a series of experiments that showed J54 impairing the activity of TLK1 and its downstream protein partners. This disrupts the entire DNA repair network of the cancer cell and accelerates DNA damage and cell death.

“Despite the immense promise shown by J54 in our experiments, it requires further exploration through molecular and cellular studies to prove its safety and effectiveness,” affirmed Dr Priya. “We aim to conduct preclinical studies to evaluate J54 in animal models and investigate its potential in combination therapies.” These experiments could confirm if inhibiting TLK1 reverses GBM’s resilience, becoming a game-changer for patients who no longer respond to standard treatment.

If met with success, the implications stretch beyond glioblastoma. Cancers from the breast to the prostate use similar repair mechanisms to evade treatment. By homing in on TLK1, researchers could attack a vulnerability shared by many aggressive tumours. “Instead of inventing stronger drugs, we could get ahead of the disease by sabotaging the cancer’s survival toolkit,” exclaimed Dr Kirubakaran as she underscored the need to create better treatment outcomes for patients.