Preclinical studies with ONC201/TIC10 and lurbinectedin as a novel combination therapy in small cell lung cancer (SCLC)

Abstract

The American Cancer Society estimates that ~15% of all lung cancers are categorized as small cell lung cancer (SCLC) with an overall five-year survival rate of less than 7%. Due to disease aggressiveness, more other malignancies, the standard of care is based on clinical efficacy rather than helpful biomarkers. Lurbinectedin is a small molecule RNA polymerase II inhibitor that binds the minor groove of DNA to induce double-strand breaks. Lurbinectedin has efficacy towards SCLC cells at sub-nM concentration and received accelerated FDA approval in 2020 for metastatic SCLC that progressed on platinum-based therapy. ONC201/TIC10 is a TRAIL pathway-inducing compound that with demonstrated clinical efficacy in H3K27M-mutated diffuse midline glioma and neuroendocrine tumors, in early phase clinical trials. We hypothesized that combining ONC201 and lurbinectedin may yield synergistic and targeted killing of SCLC cells. SCLC cell lines H1048, H1105, H1882, and H1417 were treated with ONC201 and lurbinectedin and cell viability was determined using a CellTiter-Glo assay using varying drug concentrations. Synergistic growth inhibition of SCLC cells was noted with combination of ONC201 and lurbinectedin. Induction of the integrated stress response mediator ATF4 and CHOP was observed with ONC201 and lurbinectedin along with induction of PARP cleavage indicative of apoptosis in response to cellular stress. Additionally, SCLC lines treated with the combination therapy displayed increased DNA breakage-related proteins such as phosphorylated Chk-1, Wee1 and γ-H2AX. Combination index revealed the most potent synergy occurred at the concentrations of 0.16 μM ONC201 and 0.05 nM lurbinectedin in the H1048 cell line, demonstrating highly efficient and selective killing of these tumor cells in vitro. While these therapies showed potency against the cell lines derived from SCLC patients, it is noteworthy that the combination showed significantly less toxicity to healthy human lung epithelial cells. Future studies could explore the combination of ONC201 and lurbinectedin in SCLC cell lines, SCLC patient-derived organoids, other tumor types, including in vivo studies and clinical translation.

Keywords: ONC201; SCLC; TIC10; chemotherapy; genomics; lurbinectedin.

Figure 1

Lurbinectedin & ONC201 dose-response cell viability curves with IC50 values-H1048. Lurbinectedin was administered to the wells containing approximately 5,000 H1048 SCLC cells and the cells were returned to the incubator with the treatment for 72 hours. The highest dose administered was at a concentration of 3.1087 nM. This dosage was serially diluted to a dosage of 0.0061 nM. The curve on the left was used to generate an IC50 value of 0.157 nM, indicating the concentration of lurbinectedin that kills 50% of the H1048 cells. This sub-nanomolar IC50 value indicates an exceptional specificity that this agent has for SCLC cells. The data was obtained using CellTiter-Glo (CTG) imaging technology and the kill curve and IC50 calculation were generated using Prism software by GraphPad. The IC50 value produced in this experience is well within the range of previously reported IC50 values for lurbinectedin in SCLC cells. ONC201 was administered to each well, containing approximately 5,000 H1048 SCLC cells. These cells were exposed to the ONC201 treatment for 72 hours and then cell viability was analyzed via CTG imaging technology. Using the data from the CTG, the on the right kill curve was and an IC50 value of 2.41 μM was calculated. This indicates that the concentration at which 50% of the cells was killed by ONC201. The IC50 value produced in this experience is well within the range of previously reported IC50 values for ONC201 in SCLC cells.

Figure 2

Lurbinectedin & ONC201 CTG imaging-H1048. The visual representation of lurbinectedin’s efficacy at killing SCLC cells is displayed on the left. The color panel to the right of each CTG image indicates the relative number of cells in each well, relating to the color shown on the image. It is clear that there are higher relative amounts of live cells at the lower concentrations than at the higher concentrations. These are indicated with red, yellow, and green. The higher concentrations produce wells appearing dark blue and black, indicating more dead or killed cells. ONC201’s range of killing cancer cells in the H1048 SCLC cell lines is displayed on the right, indicating the relative amount of live or dead cells in each well. It is clear that there are more live cells at the lower concentrations than at the higher concentrations in the ONC201 single agent treatment. This reduced viability at higher concentrations is the result of a possible increase in killing of cancer cells, reduced cellular proliferation, or both.

Figure 3

Lurbinectedin and ONC201 single agent treatment-H1048, 1105, and H1882 western blots. Single agent treatment in the H1048 cell line shows an increased expression in cPARP at higher concentrations when treated with lurbinectedin and ONC201, individually. This highlights the DNA damage induced by these treatments in SCLC cells as cPARP is a DNA-damage repair related protein, as more DNA is damaged, this protein is expressed accordingly. Additionally, treatment with ONC201 shows a significant increase in ATF4, underscoring the significance of ONC201’s induction of the stress-induced apoptotic pathway in these tumor cells. H1105 cell line shows an increased expression of phosphorylated Chk-1 at the stronger concentration of lurbinectedin in the single agent treatment. This correlates with the double stranded DNA breakages and associated DNA damage induced by lurbinectedin in SCLC cells. In the higher concentration single agent treatment with ONC201, ATF4 is clearly expressed in greater magnitude. This is related to ONC201’s unique mechanism of inducing the intrinsic apoptosis pathway in SCLC cells in vitro. The H1882 cell line further emphasizes the mechanisms in which both of these agents, individually, are able to effectively kill tumor cells. ONC201 shows effective mechanisms via increased expression of ATF4 and CHOP at higher doses, indicating induction of the intrinsic apoptosis pathway in these cells. Further, there is also increased expression of cPARP at the highest concentration of ONC201, indicating the agent is damaging the DNA in these cells. Similar to its effects in other SCLC cell lines, lurbinectedin shows strong expression of DNA damage-related proteins Wee1 and phosphorylated Chk1, indicating DNA damaged caused by treatment with this single agent.

Figure 4

Lurbinectedin and ONC201 combination CTG imaging-H1048, H1105, H1882, and H1417 cell lines. Lurbinectedin and ONC201 administered in combination at increasing concentrations in a 96-well plate to approximately 5,000 cells per well. As concentration increases to the right and down on the plate, it is clear that there are fewer live cells. This combination treatment is the first usage of ONC201 and lurbinectedin and its efficacy and sensitivity for SCLC cells is demonstrated in the image. A. Reduced cellular proliferation, increased cell death or a combination of the two can result in the reduced viability, displayed in the CTG imaging in the H1048 cell line. The highest concentration of lurbinectedin used was 1.5150 nM and was serially diluted to the lowest concentration of 0.0059 nM. ONC201 was serially diluted from a maximum concentration of 5.000uM to the lowest concentration of 0.156 uM. B. Lurbinectedin and ONC201 treatment in combination, suggesting effectiveness in killing tumor cells, reduced cellular proliferation, or both, replicating the trend from the H1048 cells in the H1105 cell line. C. Lurbinectedin and ONC201 treatment in combination in the H1882 cell line. D. Lurbinectedin and ONC201 combination treatment in the H1417 cell line. These results represent the trend of possible increased killing of SCLC cells, reduced cellular proliferation, or both, in vitro, at higher concentrations of the double-agent treatment. The top left well in each image serves as a control and did not receive either agent.

Figure 5

Lurbinectedin and ONC201 combination index. The combination index analysis was performed using the Combenefit [10] software revealed significant synergies at various concentrations. The most synergistic combination occurred at a lurbinectedin concentration of 0.04734 nM and an ONC201 concentration of 0.15625 μM. There were also notably high levels of synergy at the lurbinectedin concentration of 0.09469 nM and ONC201 concentration of 0.15625 μM, as well as a lurbinectedin concentration of 0.04734 nM and an ONC201 concentration of 0.3125 μM. Combenefit uses the in vitro data from the experiment and compares it to dose-responses for non-synergistic combinations [10]. The resulting figure is a mathematical representation of which doses of these drugs have the most synergistic effects in killing SCLC cells of the H1048 cell line. It is clear from this image and the data in Table 1 that there are significant synergies of these two agents, even when combined and administered at low doses of less than 0.1 nM and 0.5 μM, for lurbinectedin and ONC201, respectively.

Figure 6

DNA damage, integrated stress response activation and PARP cleavage by combination of Lurbinectedin and ONC201-H1048. The expression of DNA damage and repair-related proteins cPARP, phosphorylated Chk1 and γ-H2AX indicate a greater expression at higher concentrations of the agents. In response to cellular stress, ATF4 expression increases at the higher concentrated treatments. At higher concentrations of ONC201, and agents that induce cellular stress in cancer cells, ATF4 and other stress-induced apoptotic proteins will have increased expression, inducing death of cancer cells in the H1048 cell line. The same trend is valid for the expression of ClpP, at higher concentrations of ONC201. As ONC201 concentration increases the pro-apoptotic TRAIL pathway is induced, and the expression of the mitochondrial protein, ClpP, an electron transport chain subunit degrading-protein, is elevated. The reflection in this figure, demonstrates the mechanism by which this combination kills cancer cells in the H1048 cell line and reflects potential mechanisms for synergy at the biomolecular level.

Figure 7

DNA damage, integrated stress response activation and PARP cleavage by combination of Lurbinectedin and ONC201-H1105 and 1882. The left image reflects the western blot results from the combination treatment with lurbinectedin and ONC201 in the H1105 cell line. There is markedly elevated expression of cleaved PARP and phosphorylated Chk-1 at higher doses of the combination therapy. Further, ATF4 is upregulated in this imaging, indicating ONC201 working via the induction of the apoptotic TRAIL pathway. This imaging at 24 hours is early enough in the SCLC cell elimination process to see the expression of these DNA damage-related and apoptosis related proteins increasing in response to exposure to lurbinectedin and ONC201 in the H1105 cell line. The results of the western blot analysis from lurbinectedin and ONC201 combination treatment in the H1882 SCLC cell line, in the right image, reflect the same pattern of pharmacologically induced DNA damage in the SCLC cells as well as increased expression of proteins associated with the pro-apoptotic TRAIL pathway. It is clear that higher dosages of this combination treatment show larger bands, higher levels of expression, of proteins related to these cell-killing mechanisms. This is consistent with the mechanisms in the other cell links which also show an upregulation in TRAIL-related proteins such as ATF4 and CHOP as well as proteins related to the cellular response to DNA damage, such as phosphorylated Chk-1 and cleaved PARP.

Figure 8

Lurbinectedin and ONC201 combination treatment-CTG imaging-healthy lung epithelial cells. This image reflects the claims that ONC201 and Lurbinectedin selectively target malignant cells in SCLC and spare healthy epithelial cells from their toxic effects. After 24 hours of treatment, CTG analysis reveals that there are quantifiable numbers of live cells in each well of the plate at every concentration, including the highest concentrations of treatment, which left zero cells alive when used in the malignant SCLC cell lines: H1048, H1105 and H1882. This further validates the clinical claims from patients who prefer lurbinectedin due to its limited side effect profile when compared to traditional chemotherapies used in first-line treatment of SCLC.