Janus kinases as targets for small molecule inhibitors

Doctoral thesis (2010)

Many cytokines transduce signals by employing receptor/Janus kinase (Jak) complexes that, once activated, promote phosphorylation of several signaling proteins such as STAT transcription factors. The Jak ... [more ▼]

Many cytokines transduce signals by employing receptor/Janus kinase (Jak) complexes that, once activated, promote phosphorylation of several signaling proteins such as STAT transcription factors. The Jak/STAT pathway is normally tightly modulated by regulatory mechanisms preventing its over-activation. However, a number of genetic alterations in Jak kinase genes have been found in myeloproliferative neoplasms and leukemia that render the Jaks hyperactive and cytokine-independent. In the present thesis pharmacologic and genetic approaches were applied to study aspects of both the cytokine-dependent and -independent activation of the Jak/STAT pathway. We applied a chemical genetics approach to Janus kinases and generate analogue-sensitive Jak1 and Jak2 mutants. With these tools we could for the first time specifically inhibit either Jak1 or Jak2 and investigate their relative contributions in IFN signaling. Single inhibition of either Jak by an inhibitor analogue showed differential effects on IFN-mediated STAT1 phosphorylation, Jak2 inhibition having a stronger suppressive effect. Furthermore, we identified STAT1 and IRF1 as genes, whose increased expression upon IFN stimulation strongly depends on active Jak2 but not on Jak1. Most of the IFN-regulated genes, however, depend on both Jak1 and Jak2 activity. Exploiting this quick and reversible pharmacological inhibition of analogue-sensitive kinases we address the temporal requirement of Jak activity and the importance of different phases of the IFN response. Moreover, we further characterized constitutively active Jak2 mutants and show that SOCS proteins seem to be directly involved in their proteasome-mediated degradation. There is an increasing interest in developing small molecule inhibitors targeting Jaks and Jak mutants. We show that Erlotinib, an inhibitor targeting EGFR which is already used in the clinic to treat lung and pancreatic cancer, can have a beneficial additional effect if combined with a Jak-specific inhibitor on cell growth suppression and apoptosis of Jak2-V617F- or Jak3-A572V-positive leukemic cells. Erlotinib, in contrast to Jak inhibitors targeting Jaks directly affects the eIF2/ATF4 pathway in leukemic cells. This is of general interest since Erlotinib treatment in conjunction with other kinase inhibitors could be an interesting approach also in other kinase-dependant leukemias (e.g. BCR-ABL-positive CML). [less ▲]

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.

in Oncogene (2009), 28(34), 3069-80

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS ... [more ▼]

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia. [less ▲]