- Letter to the Editor
- Open Access
BCL-XL PROTAC degrader DT2216 synergizes with sotorasib in preclinical models of KRASG12C-mutated cancers
Journal of Hematology & Oncology volume 15, Article number: 23 (2022)
KRAS mutations are the most common oncogenic drivers. Sotorasib (AMG510), a covalent inhibitor of KRASG12C, was recently approved for the treatment of KRASG12C-mutated non-small cell lung cancer (NSCLC). However, the efficacy of sotorasib and other KRASG12C inhibitors is limited by intrinsic resistance in colorectal cancer (CRC) and by the rapid emergence of acquired resistance in all treated tumors. Therefore, there is an urgent need to develop novel combination therapies to overcome sotorasib resistance and to maximize its efficacy. We assessed the effect of sotorasib alone or in combination with DT2216 (a clinical-stage BCL-XL proteolysis targeting chimera [PROTAC]) on KRASG12C-mutated NSCLC, CRC and pancreatic cancer (PC) cell lines using MTS cell viability, colony formation and Annexin-V/PI apoptosis assays. Furthermore, the therapeutic efficacy of sotorasib alone and in combination with DT2216 was evaluated in vivo using different tumor xenograft models. We observed heterogeneous responses to sotorasib alone, whereas its combination with DT2216 strongly inhibited viability of KRASG12C tumor cell lines that partially responded to sotorasib treatment. Mechanistically, sotorasib treatment led to stabilization of BIM and co-treatment with DT2216 inhibited sotorasib-induced BCL-XL/BIM interaction leading to enhanced apoptosis in KRASG12C tumor cell lines. Furthermore, DT2216 co-treatment significantly improved the antitumor efficacy of sotorasib in vivo. Collectively, our findings suggest that due to cytostatic activity, the efficacy of sotorasib is limited, and therefore, its combination with a pro-apoptotic agent, i.e., DT2216, shows synergistic responses and can potentially overcome resistance.
To the Editor,
KRAS mutations are the most common drivers in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic cancer (PC) . While KRASG12C inhibitors including sotorasib have shown tumor responses in a subset of NSCLC, there was reduced activity in CRC patients. To enhance its efficacy, sotorasib has been evaluated in preclinical studies using different combinations [2,3,4]. These combinations, however, are mostly cytostatic, limiting the potential for clinical benefit.
BCL-XL is an anti-apoptotic protein that belongs to the BCL-2 family and is an important therapeutic target in multiple cancers. However, targeting BCL-XL with conventional inhibitors causes severe thrombocytopenia, limiting their clinical use [5,6,7]. Recently, our group has reported platelet-sparing targeting of BCL-XL by proteolysis targeting chimeras (PROTACs) exemplified by DT2216. DT2216 has shown promising antitumor activities in BCL-XL-dependent hematologic cancers as a single agent therapy and in multiple solid tumors when combined with conventional chemotherapy [8,9,10]. Here, we hypothesize that combining sotorasib with DT2216 could be safer and synergistic, because BCL-XL is overexpressed in KRAS-mutated tumors .
We found that 1 µM of DT2216 can completely deplete BCL-XL in different KRASG12C-mutated cancer cell lines (Additional file 1: Fig. 1a-d); therefore, we used this concentration for in vitro experiments. In our study, sotorasib showed heterogeneous effects against KRASG12C-mutated cancer cell lines (Fig. 1a; Additional file 1: Fig. 2a). Moreover, sotorasib caused only partial reduction in viability in the sensitive cell lines (referred to as partially sensitive cell lines). Interestingly, a combination of sotorasib and DT2216 synergistically reduced viability of partially sensitive cell lines, as well as enhanced inhibition of colony formation and apoptosis induction compared to sotorasib monotherapy (Fig. 1a–d). We further evaluated whether or not the inhibition of other BCL-2 anti-apoptotic proteins could enhance the efficacy of sotorasib. We found that the inhibition of BCL-XL, but not BCL-2 or MCL-1, can sensitize all the three partially sensitive cell lines to sotorasib treatment (Additional file 1: Fig. 2b). Moreover, non-KRASG12C-mutated cell lines did not respond to sotorasib treatment alone as well as in combination with DT2216 (Additional file 1: Fig. 2c). Of note, the synergistic effect of sotorasib + DT2216 combination was significantly abrogated in the presence of a pan-caspase inhibitor (QVD-OPh), indicating caspase-mediated apoptosis (Additional file 1: Fig. 3). In addition, DT2216 could not synergize with a MEK inhibitor (selumetinib) in the KRASG12C-mutated cancer cell lines that did not respond to sotorasib + DT2216 combination (Additional file 1: Fig. 4). These results suggest that DT2216 can potently enhance the efficacy of sotorasib in KRASG12C-mutated cancer cells which are partially sensitive to sotorasib monotherapy.
Next, we elucidated the mechanism involved in the DT2216 + sotorasib synergistic activity. DT2216 co-treatment with sotorasib was not able to enhance or prolong the inhibition of KRAS signaling (Additional file 1: Fig. 5a-h; Additional file 1: Fig. 6). Therefore, we hypothesized that sotorasib might induce apoptotic priming through the stabilization of BH3-only pro-apoptotic proteins (e.g., BIM, BMF and PUMA). We observed a concentration-dependent upregulation of BIM and BMF after sotorasib treatment in all the partially sensitive cell lines, while PUMA was also upregulated in MIA PaCa-2 and SW837 (Fig. 1e–g; Additional file 1: Fig. 7a-d and 8a-d). Sotorasib or sotorasib + DT2216 had no considerable effect on other BCL-2 proteins (Additional file 1: Fig. 7a-d). Further, sotorasib + DT2216 combination led to a pronounced increase in cleaved caspase-3 and cleaved PARP levels in partially sensitive cell lines indicating apoptosis induction (Additional file 1: Fig. 7a-c). We observed a decrease in p-BIM (S69) levels after sotorasib treatment (Additional file 1: Fig. 9a, b), which might be attributed to BIM stabilization as ERK activation is known to induce BIM (S69) phosphorylation and degradation (12). In addition, sotorasib led to a concentration-dependent upregulation of BCL2L11 (BIM coding gene) (Additional file 1: Fig. 9c). Next, we found that sotorasib selectively induces BCL-XL interaction with BIM, which was disrupted upon BCL-XL degradation with DT2216 (Fig. 1h-j). These results suggest that sotorasib induces apoptotic priming that can be exploited by DT2216 to induce apoptosis in KRASG12C-mutated cancer cells.
Finally, we investigated the efficacy of sotorasib + DT2216 combination in mouse xenografts. As expected, DT2216 alone had no significant effect on tumor growth. The DT2216 + sotorasib combination led to significant tumor inhibition compared to sotorasib monotherapy (Fig. 2a–f). The combination treatment was quite safe as indicated by no significant change in mouse body weights, as well as no clinically significant decrease in blood cell counts was seen (Additional file 1: Fig. 10a-c; Additional file 1: Fig. 11a, b). We also confirmed BCL-XL degradation and KRAS engagement with DT2216 and sotorasib treatments, respectively (Fig. 2g–i; Additional file 1: Fig. 12a). In addition, sotorasib-mediated inhibition of ERK was associated with BIM accumulation in MIA PaCa-2 xenograft tumors leading to an increase in cleaved caspase-3 and cleaved PARP in combination-treated tumors (Additional file 1: Fig. 12b, c). Further, IHC staining showed a considerable decrease in Ki67 and a significant increase in cleaved caspase-3 in combination-treated H358 tumors (Additional file 1: Fig. 12d), which was consistent with tumor growth inhibition (Fig. 2a, d).
In conclusion, our studies show that DT2216 enhances the therapeutic efficacy of sotorasib which warrants clinical testing of this combination, particularly in KRASG12C-mutated CRC patients who otherwise derive minimal benefit from sotorasib monotherapy.
Availability of data and materials
All data generated or analyzed during this study are included in this published article or its supplementary information files. The raw datasets are available from the corresponding authors on reasonable request.
Non-small cell lung cancer
Receptor tyrosine kinase
- BCL-XL :
B-cell lymphoma extra-large
Proteolysis targeting chimera
Moore AR, Rosenberg SC, McCormick F, Malek S. RAS-targeted therapies: Is the undruggable drugged? Nat Rev Drug Discov. 2020;19(8):533–52.
Canon J, Rex K, Saiki AY, Mohr C, Cooke K, Bagal D, et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature. 2019;575(7781):217–23.
Ryan MB, Fece de la Cruz F, Phat S, Myers DT, Wong E, Shahzade HA, et al. Vertical pathway inhibition overcomes adaptive feedback resistance to KRAS. Clin Cancer Res. 2020;26(7):1633–43.
Misale S, Fatherree JP, Cortez E, Li C, Bilton S, Timonina D, et al. KRAS G12C NSCLC models are sensitive to direct targeting of KRAS in combination with PI3K inhibition. Clin Cancer Res. 2019;25(2):796–807.
Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ellis S, et al. Programmed anuclear cell death delimits platelet life span. Cell. 2007;128(6):1173–86.
Zhang H, Nimmer PM, Tahir SK, Chen J, Fryer RM, Hahn KR, et al. Bcl-2 family proteins are essential for platelet survival. Cell Death Differ. 2007;14(5):943–51.
Schoenwaelder SM, Jarman KE, Gardiner EE, Hua M, Qiao J, White MJ, et al. Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets. Blood. 2011;118(6):1663–74.
Khan S, Zhang X, Lv D, Zhang Q, He Y, Zhang P, et al. A selective BCL-X L PROTAC degrader achieves safe and potent antitumor activity. Nat Med. 2019;25(12):1938–47.
He Y, Koch R, Budamagunta V, Zhang P, Zhang X, Khan S, et al. DT2216-a Bcl-xL-specific degrader is highly active against Bcl-xL-dependent T cell lymphomas. J Hematol Oncol. 2020;13(1):95.
Zhang X, Thummuri D, Liu X, Hu W, Zhang P, Khan S, et al. Discovery of PROTAC BCL-X L degraders as potent anticancer agents with low on-target platelet toxicity. Eur J Med Chem. 2020;192:112186.
Kasper S, Breitenbuecher F, Reis H, Brandau S, Worm K, Köhler J, et al. Oncogenic RAS simultaneously protects against anti-EGFR antibody-dependent cellular cytotoxicity and EGFR signaling blockade. Oncogene. 2013;32(23):2873–81.
Luciano F, Jacquel A, Colosetti P, Herrant M, Cagnol S, Pages G, et al. Phosphorylation of Bim-EL by Erk1/2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. Oncogene. 2003;22(43):6785–93.
This study was supported in part by US National Institutes of Health (NIH) Grant R01 CA242003 (D.Z. & G.Z.).
Ethics approval and consent to participate
All the animal procedures were performed in accordance with the rules of the IACUC at the University of Florida.
Consent for publication
S.K., P.Z., D.T., G.Z. and D.Z. are inventors of two patent applications for use of BCL-XL PROTACs as senolytic and antitumor agents. G.Z. and D.Z. are co-founders of and have equity in Dialectic Therapeutics, which develops BCL-XL/2 PROTACs to treat cancer.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Khan, S., Wiegand, J., Zhang, P. et al. BCL-XL PROTAC degrader DT2216 synergizes with sotorasib in preclinical models of KRASG12C-mutated cancers. J Hematol Oncol 15, 23 (2022). https://doi.org/10.1186/s13045-022-01241-3
- Drug resistance