TYK2 inhibition for CALR-mutated myeloproliferative neoplasms: a novel selective treatment approach

Genetic mutational drivers of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are mutations in the genes of Janus kinase (JAK)2, calreticulin (CALR), and the thrombopoietin receptor myeloproliferative leukemia (MPL). JAK2V617F-mutant cells are shown to be more sensitive to interferon alpha (IFNα) treatment compared with CALR-mutant (CALR^mut) cells due to greater phosphorylation of JAK1 and signal transducer and activator of transcription (STAT)1 in JAK2V617F‑mutant cells.

Stimulation of IFNα elicits phosphorylation of tyrosine kinase 2 (TYK2) and JAK1, which are known to be critical regulators of signal transduction pathways. TYK2 and JAK1 are associated with IFNα receptor-1 and -2, respectively, and activate the downstream signaling pathways, which in turn recruits specific STAT proteins that translocate to the nucleus and regulate gene expression. Inhibition of TYK2 has shown promising clinical efficacy and favorable safety profiles in auto-inflammatory diseases. However, there is limited evidence on the role of TYK2 in the survival and IFNα response to JAK2 in MPN cells versus CALR^mut MPN cells.

During the European Hematology Association (EHA)2021 Virtual Congress, Rebecca Lemanzyk¹ presented the findings from their study on the role of TYK2 in MPN cells. The MPN Hub is pleased to present the key findings here.

Aims and methods

The aims and methods of the study included:

• Investigating TYK2 phosphorylation (p-TYK2) in 32D^MPL CALR 52-base pair deletion (CALR^del52) and JAK2V617F cells.
• Generating TYK2-knockout (KO) in 32D^MPL JAK2V617F or CALR^del52 cells using a CRISPR/Cas9 system.
• Investigating the role of TYK2 in IFNα response on cell viability, downstream JAK-STAT signaling, and gene expression using CRISPR/Cas9 system-edited 32D^MPL clones.
• Investigating the effects of the TYK2 inhibitor deucravacitinib in 32D^MPL-mutant cells, and in primary MPN peripheral blood mononuclear cells (PBMCs) from patients with polycythemia vera, essential thrombocythemia (ET), or primary myelofibrosis (PMF), using colony formation unit assays.

Results

Increased p-TYK2 in 32D^MPL CALR^del52 cells

p-TYK2 was increased in the 32D^MPL CALR^del52-mutant cells compared with 32D^MPL JAK2V617F-mutant cells and empty vector cells (p < 0.001). Whereas, phosphorylated‑JAK2 (p-JAK2) was increased in 32D^MPL JAK2V617F cells compared with 32D^MPL CALR^del52-mutant cells.

Selection against TYK2 KO in 32D^MPL CALR^del52 cells

Genotyping of initial clones demonstrated an altered TYK locus either homozygously or heterozygously. Base pair at 400 represented TYK2 KO cells and a raised base pair suggested wildtype (WT) cells. However, p‑TYK2 was retained in the initial clones as observed with western blotting. Regenotyping with new clones from the same cell lines demonstrated only WT remained at Day 21.

Successful generation of p‑TYK2-deficient 32D^MPL JAK2V617F clones

Using western blotting, 32D^MPL JAK2V617F clones demonstrated the expression of TYK2. However, clones 19 and 43 were p‑TYK2-deficient, while clones 2 and 6 only showed a faint line for the p‑TYK2 band, suggesting failure to generate TYK2 KO cells in CALR^del52-mutant cells, and the importance of TYK2 in CALR-mutant cells. Clones 19 and 43 both showed the same heterozygous alterations of the TYK2 locus, with one allele harboring an in-frame deletion and the other harboring a frameshift mutation leading to a premature stop codon.

IFNα response is p‑TYK2-dependent

Clones treated with IFNα demonstrated a dose-dependent decrease in metabolic activity in the parental bulk and in the WT clones 37 and 42, compared with an increased metabolic activity in p‑TYK2-deficient clones 19 and 43. Significant induction of apoptosis was observed on treatment with IFNα in all cell clones. However, there were no significant differences between p‑TYK2-deficient and TYK2 WT 32D^MPL JAK2V617F cells.

Induction of IFNα target gene expression is p-TYK2-dependent

IFNα-treated parental bulk and WT cells showed a significant expression of Mx1 and Stat1 genes. However, no significant gene expression was observed for the p‑TYK2-deficient clone 43.

Reduced downstream signaling in p‑TYK2 deficient clones

p-TYK2-deficient clones, 19 and 43, treated with IFNα, showed a reduction of p‑STAT3, while STAT1 expression was increased. Although there was similarity in the p‑STAT1 band in all clones, overexpression of STAT1 lead to reduction in p‑STAT1 in p‑TYK2-deficient clones

32D^MPL CALR^del52 cells are sensitive to inhibition of TYK2

The TYK2 inhibitor deucravacitinib demonstrated a significant reduction in metabolic activity in CALR^del52-mutant cells even at low concentrations of 0.5 µM, but this was not observed in JAK2V617F-mutant cells. p-TYK2 was present only in CALR^del52-mutant cells but was ablated with deucravacitinib. Deucravacitinib did not show any effects on p-JAK2; however, it reduced p‑STAT3 and p‑STAT5 in CALR^del52-mutant cells.

Colony growth and allele burden are reduced in primary samples from patients with ET or PMF

The TYK2 inhibitor deucravacitinib had less of an effect on PBMCs from patients with polycythemia vera compared with PBMCs from patients with ET or PMF. No significant differences were observed between CALR^del52-mutant cells and JAK2V617F-mutant cells from patients with ET or PMF. CALR^del52 positive patients showed a reduced allele burden with TYK2 inhibition.

Conclusion

The findings demonstrated that TYK2 was required to deliver an adequate response to IFNα. TYK2 played an important role in CALR^del52-mutant cells but not in JAK2V617F-mutant cells, and it demonstrated the potential to be a novel treatment approach in CALR^mut MPN. Future studies should investigate the interaction of TYK2 and MPL, establish successful generation of KO TYK2 in cell lines, and validate the findings in vivo studies.