CALR-Mutant Myeloproliferative Neoplasms Induced by MPL Downstream Activation

Myeloproliferative neoplasms (MPNs) are a group of rare blood cancers that results from excessive proliferation of hematopoietic stem cells; these conditions are often chronic and harbor the potential to transform into acute myeloid leukemia.

Some patients with MPNs are known to experience a mutation of calreticulin (CALR), a protein that can bind to other misfolded proteins and inhibit their degradation. The mutated CALR is known to activate thrombopoietin receptor, another protein, which has been linked with the development of many cancers.

While previous research has established that activation of the thrombopoietin receptor molecules (MPL) occurs in the disease, the molecular mechanism behind the activity of mutant CALR has remained a mystery—researchers from Juntendo University have supplied another piece to the puzzle of MPNs with the discovery that the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPNs.

In a recent interview at ASCO 2018 Ruben Mesa, MD, recently told Rare Disease Report^® that one of the biggest challenges regarding MPNs is gaining an understanding of how they develop and why they progress.

“I’d say we learned more about the genetic pieces, but it’s not fully mapped out,” he said. “What I share with patients [is that] it’s a bit like a jigsaw puzzle that has 500 pieces, but we only have 200 on the board. We understand some parts—there are some parts of the picture that are fleshed out—but they’re not all there.”

To get some answers, the researchers first drew from previously established data which indicated that mutant CALR activates MPL and a set of signaling molecules, one of which is known as JAK2, and thus, induces cancer.

This grounding data included findings from a past study published in Blood. The research yielded 2 main takeaways: mutant CALR induces thrombopoietin (TPO)-independent growth in the human megakaryocytic cell line UT-7/TPO and mutant CALR binds to the TPO receptor, inducing phosphorylation of JAK2 and activates downstream signaling.

With this knowledge, the research team formed their hypothesis for the current study, that a particular association between 2 thrombopoietin receptor molecules is mediated by the interaction between mutant CALRs.

Mutant CALR interact to create homomultimeric complexes, which are clusters of identical proteins. “This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN,” the study abstract reads. Non-mutant CALRs, referred to as wild-types, do not do this.

Through the use of a competition assay, the researchers were able to demonstrate that the formation of mutant CALR homomultimers is needed for the binding and activation of MPL. Based on their observations, the researchers postulate that actual MPL activation occurs when homomultimeric mutant CALR interact at the same time with 2 MPL molecules, which works to induce the formation of JAK2 and the subsequent biochemical signaling pathways during MPN development.

The researchers concluded, based on their observations, that inhibition of the intermolecular interaction can potentially be used to prevent tumor formation; however, more research is needed.

Though the team’s findings are unable provide answers for all of the pressing questions surrounding MPN disease mechanisms, they did posit that they could be potentially be used to inform the development of more effective treatments.

"Although more detailed molecular and structural analyses are required to understand the mechanism behind MPL activation by mutant CALR proteins,” the researchers conclude in a recent statement, “our findings shed light on MPN pathogenesis and provide support for the development of novel therapeutic strategies against MPNs with mutant CALR proteins."