Team finds that blocking CHK2 and ERK increases apoptosis and inhibits cell proliferation.
Researchers suggest that inhibiting both ERK and the checkpoint protein CHK2 could provide a new approach to treating diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma. A University of Maryland School of Medicine-led team has found elevated levels of both proteins in human DLBCL, and in addition demonstrated a physical interaction between them.
The scientists also showed that administration of an ERK inhibitor enhances the antitumor activity of CHK2 inhibition in both a human DLBCL xenograft model as well as primary human DLBCL cells. Ronald B. Gartenhaus, M.D., and colleagues, report their findings in Nature Communications. Their paper is titled “Functional and molecular interactions between ERK and CHK2 in diffuse large B-cell lymphoma.”
The kinase CHK2 is a key mediator of the DNA damage checkpoint that responds to DNA double-strand breaks (DSBs). Recent studies have identified defects in the protein in a range of sporadic malignancies and shown that aberrations in CHK2 predispose to several types of hereditary carconima. The extracellular signal-regulated kinases 1 and 2 (ERK1/2), meanwhile, regulate cell proliferation and survival, and deregulation of ERK signaling is associated with cell proliferation and survival. Previous work by the University of Maryland team showed that ERK inhibition induced the apoptosis of human DLBCL cells and had marked antitumor activity in human DLBCL xenograft models, implying a potential application of ERK targeting for DLBCL therapy.
There is, however, little data on CHK2 and ERK signaling in lymphoid malignancies, Dr. Gartenhaus’ team admits, which prompted them to investigate any potential therapeutic effects of combining ERK and CHK2 inhibitors against DLBCL.
The team first carried out immunohistochemical analysis of samples from 50 primary DLBCL specimens and 24 reactive lymph nodes. They found increased levels of ERK1/2 and CHK2 in 100% of germinal center B-cell (GCB) DLBCL samples. ERK1/2 levels in DLBCL appeared to correlate strongly with CHK2 protein levels, “supporting their value as potential therapeutic targets,” the authors write. “These data indicate that increased coexpression of ERK1/2 and CHK2 may be associated with the development of DLBCL.”
Because previous studies have shown that inhibiting CHK2 expression attenuates DNA damage-induced cell cycle checkpoints and increases apoptotic activity in cancer cells, the researchers moved on to investigate whether inhibiting CHK2 would also induce apoptosis in DLBCL cells. Administration of CHK2 inhibitor II to SUDHL4, SUDHL6, and Farage cells resulted in apoptosis of less than 30% of cells. These results mirror those of previous studies by the team, which demonstrated that less than 30% of DLBCL cells underwent apoptosis when exposed to an ERK inhibitor. In contrast, subjecting OCI-LY3, Farage, SUDHL4, and SUDHL6 cells to a combination of CHK2 inhibitor II and the ERK inhibitor resulted in substantially higher levels of apoptosis compared with treatment using either drug alone.
Interestingly, the two proteins appeared to physically associate: the researchers detected reciprocal co-immunoprecipitation of endogenous ERK1/2 with CHK2 in Farage, SUDHL5, and SUDHL6 cells. In vitro glutathione S-transferase (GST) fusion protein pull-down assays separately showed that the GST–ERK2 fusion protein was able to pull down CHK2.
Moreover, this physical interaction between ERK and CHK2 was blocked by CHK2 inhibition. Given that CHK2 inhibitor II blocks CHK2 phosphorylation at Thr68 (phosphorylation at Thr68 serves as a surrogate marker for CHK2 activation) it appears that Thr68 phosphorylation is involved in the physical interaction between ERK and CHK2, the authors point out.
Indeed, while wild-type CHK2 expressed in HEK293 and HeLa cells co-immunoprecipitated with ERK2, a T68A mutant CHK2 expressed in these cells did not. GSK pull-down experiments similarly showed that the GST-ERK2 protein can pull down wild-type CHK2, but not the T68A mutant.
The interaction of CHK2 with ERK suggests that the two proteins are involved in reciprocal regulation, and ERK may be involved in CHK2-mediated DNA damage responses, the researchers note. “This is also supported by our observation that ERK inhibitor potentiates CHK2 inhibitor-induced cell apoptosis.” However, what wasn’t yet clear was how these two proteins regulate each other and why co-treatment with an ERK and CHK2 inhibitor increases apoptosis.
In a series of experiments designed to investigate this phenomenon further, they found that while CHK2 inhibitor II decreases CHK2 phosphorylation at Thr68, it also increases ERK phosphorylation in SUDHL6, Farage, and OCI-LY3 cells. A similar result was obtained when CHK2 was genetically modified to inhibit Thr68 phosphorylation. In concurrence, the researchers showed that the T68A mutant activated ERK, whereas wild-type CHK2 failed to do so.
It thus appears that CHK2-ERK interaction negatively regulates ERK, and inhibiting CHK2 leads to increased activation of ERK. The benefits of inhibiting CHK2 as an approach to treating DLBCL cells may therefore be improved significantly by inhibiting ERK as well.
Moving on to test dual inhibition of CHK2 and ERK in DLBCL tumors in vivo, the team established SUDHL6 DLBCL xenografts in 28 SCID mice, and treated cohorts of these animals with either the CHK2 inhibitor II, the ERK inhibitor, or dual inhibition. Single inhibitor therapy modestly inhibited tumor growth, but combined therapy resulted in statistically significant suppression of tumor growth. Significantly, the dual therapy caused a striking increase in H2AX phosphorylation and PARP cleavage, indicative of increased double-stranded DNA breaks and apoptosis. Immunohistochemical analysis of tumor sections showed that co-administration of the ERK and CHK2 inhibitors also markedly reduced expression of the cell proliferation marker Ki67. Encouragingly, dual treatment demonstrated no lethal toxicity, significant weight loss, or any other gross abnormalities among treated animals, and microscopic examination of mouse organs showed no evidence of tissue damage.
Treating suspensions of confirmed DLBCL lymph node biopsy cells using the combined inhibitor therapy similarly resulted in enhanced levels of apoptosis compared with either inhibitor alone. In addition, combined treatment with the ERK and CHK2 inhibitors dramatically reduced cells’ viability whereas either inhibitor administered individually had much less effect.
“Our study demonstrates the mechanistic basis for the therapeutic targeting of DLBCLs with inhibition of both ERK and CHK2 and provides a rationale for using this combinatorial therapy,” the authors conclude.
They admit the finding that inhibition of CHK2 activity leads to activation of ERK, which may preserve cell survival and proliferation, was unexpected. “This provides a plausible explanation for the modest induction of cell apoptosis by CHK2 inhibitor and suggests that activation of ERK signaling pathway may represent a compensatory response to CHK2 inhibition,” they write. “In other words, functional CHK2 may be required for cells to survive the basal level of genomic instability known to be present in many malignancies. However, the resulting ERK activation in response to CHK2 inhibition provides a potent survival signal that attenuates the apoptotic drive.”