War against the Clones.
by Zhenya Senyak
It took a small army of investigators, mathematicians, lab rats, geneticists, and programmers to discover the tactics used by the Mutant Clones to withstand every effort to destroy them.
In 2005, five labs independently discovered the presence of the JAK2 mutation common to most MPN patients. Over the next seven years , we in the MPN community have taken heavy casualties, dead and wounded, at the hands of a crafty, relentless enemy that despite intensive bombardment with medical technology has shown itself surprisingly resilient and capable of counter-attack.
Now, a breakthrough. A group of scientists across multiple institutions — funded in part by the NCI, the LLS and MPN Research Foundation – has cracked one part of the enemy code that should allow us to mount a new offensive with improved tactics and the promise of more effective weapons. This is a commentary on their report as it appeared in Nature, last week.
Nature (2012) doi:10.1038/nature11303 Received 11 May 2011 Accepted08 June 2012 Published online 22 July 2012
Two takeaway points:
(1) Resistance to Jakafi therapy is reversible. Withdrawal from JAK2 inhibitor therapy results in renewed sensitivity to the drug’s ability to interrupt expression of the JAK2 clone.
(2) Even those cells that persist in generating MPN blood products despite JAK2 inhibition still depend on the JAK2 protein expression. Thus treatments that inhibit or degrade JAK2 like (Heat Shock Protein 90 or HDAC –histone deacetylase inhibitors) are likely to benefit MPN patients.
A milestone publication
The Letter published in the journal Nature last week, provides some answers to vexing myelofibrosis question and holds out hope that exhausted benefits from Jakafi therapy might be revived after a short interruption.
Filled with dense scientific terminology and technical references, the Letter Heterodimeric JAK-STAT activation as a mechanism of persistence ot JAK2 inhibitor therapy has all the earmarks of a scientific breakthrough. The findings it reports will likely be translated into drug development and new therapy protocols.
The Letter both advances our scientific understanding of the complex processes involved in producing MPNs and provides practical and immediate avenues for drug exploration and therapeutic protocol
Ross Levine, one of the investigators sharing in the discovery of the JAK2 mutation in 2005, is lead author. He and his associates at Memorial Sloan Kettering, along with investigators from MD Anderson, Howard Hughes, the Broad Institute and Harvard Medical School explored a well-observed phenomenon: Some cells requiring JAK2 signalling to produce one or more blood lines continue to churn out MPN cells despite the presence of JAK2 inhibitors. That phenomenon helped spark the research that led to these discoveries.
In 2005 a single mutation — JAK-V617F — was found to be common in many MPNs, a crossed signal arising during reproduction that produced a mutation in an enzyme critical in signaling blood production. Because of their role in hematopoiesis, the tyrosine kinases – TYK2, JAK1, JAK2, and JAK3 – are of special interest to those tracking the causes of blood cancers. The focus has been on JAK2 the mutated enzyme showing up in almost all cases of PV and most MPNs.
While the signaling pathways and biochemistry are complex, the process itself is fundamentally simple. Phosphorylation, the addition of a phosphate to a protein, activates a protein, turns it on. The JAK2 mutation created an enzyme that phosphorylates itself, that is always active whether or not the body is signaling for production of a specific blood element. The result can be proliferation of platelets or erythrocytes (red blood cells) . The excessive blood production produces all the symptoms common to MPNs along with fibrosis in the overworked marrow.
With this information in hand, the drug companies set about designing and testing molecules that would inhibit or shut down the mutated JAK2 enzyme and end proliferation. Through lab work and extensive clinical trial they both succeeded…and didn’t. One JAK2 inhibitor, INCB18424, ruxolitinib, proved to be effective in clinical trial, in many cases, for prolonged periods of time. And then not work at all. Despite inhibition, hematopoietic precursor cells persistently turned out MPN cells. It defied logic
For some reason, despite the effective inhibition of JAK kinases and the reduction of patient symptoms, the MPN disease burden continued unabated.
Based on these observations, investigators looked at these Jakafi-resistant cells in light of the central role of the JAK-STAT pathway. JAK-STAT is the main transducing pathway downstream of growth factors EPO and Thrombopoietietin. The most common disruption in the JAK-STAT pathway is the JAK2 v617F mutation, the signature MPN mutant clone.
Hematopoiesis requires cytokine signalling mediated by tyrosine kinases, the JAKs, and a downstream protein within the cell, the STAT (Signal Transducters and Activators of Transcription). Once activated the STAT dimer enters the cell nucelus and docks at the appropriate DNA point to pass on the signal and get the blueprint of a target gene. In humans, there are four JAK enzymes and five STAT proteins. It takes a pair of each – a dimer – to get the process going.
Levine et.al. reported it is not only the JAK2 enzyme that can provide the necessary kickstart, or a team of two JAK2 enzymes – a homodimer. Other JAK enzymes – like TYK1 and JAK1 – can join with JAK2 to form a heterodimer — effective in activating the STAT proteins.
Calling on other JAK kinase clan members to stand in for the inhibited JAK2 enzyme has proven an effective strategy for the mutant clone. The resulting heterodimer complex is persistent enough that JAK cells become resistant to the effects of JAK inhibitors.
The good news: While activating JAK-STAT signaling with the help of other JAKs can maintain or increase the disease burden, the process is reversible. Remove the JAK2 inhibition and the cell becomes resensitized, the patient once again amenable to inhibition therapy.
“We then investigated whether persistence of JAK2 inhibitors was reversible. We removed INCB18424…for 2-4 weeks. This led to JAK inhibitor resensitization. Resensitized JAK2 cells were sensitive to all three JAK inhibitors, suggesting that patients with MPN may respond to retreatment or to a different JAK2 inhibitors after a brief withdrawal of treatment.“
These heterodimeric cells that persist in producing MPN cells in the presence of inhibitors,are nevertheless “still dependent on JAK2 protein expression,” suggesting continued therapeutic use of such inhibitors in face of persistence.
The answer to some vexing questions…
This research provides important clues as to why, once Jakafi demonstrates its effectiveness at some point, it simply becomes ineffective in controlling myelofibrosis symptoms. To put it another way, MPN cells survive despite chronic inhibition of JAK kinases enabling MF to overwhelm the therapy.
This is not, say the authors, a case of acquired drug resistance due to mutation or some other factors. The inablility of JAK inhibitors to decrease the burden of mutated cells is “due to persistent growth and signalling” despite JAK2 kinase inhibition. That signaling is possible because other JAK kinases are recruited to complete the formation of the dimer necessary to produce the mutant clone. These heterodimers both expand the clone’s staying power…and present promising new targets for drug developers.
This recruitment and survival strategy is completely reversible . Once JAK inhibition ceases, the cells recover their sensitivity to JAK2 inhibition after a brief interlude and continuation of renewed therapy is likely to be effective. ”I think, ” says Mayo Clinic’s Dr. Ruben Mesa, ” retrying (Jakafi) in patients who were on ruxo, and now off, is a very fair question based on this paper.”
Then there’s the question of why a JAK2 inhibitor would be equally effective in managing myelofibrosis symptoms in patients who do not have the JAK2 mutation. That’s about half of all MF patients. The research reported on in the Levine Nature letter does not address this question as all cells, human and murine, studied were positive for the mutation.
However, now that it has been established that heterodimerization is responsible for the persistence of MPN cells in the presences of inhibition, it is only a small leap to posit the possibility that other kinases disrupt the JAK STAT pathway in ways that JAK inhibition resolves. And, since proliferation and differentiation of all hemapoietic cells depend on JAK2 expression, inhibition of this kinase would seem capable of disrupting downstream signaling .
That’s speculation. Here are some immediate therapeutic implications of this work.
The Levine group started this work while all JAK2 inhibitors were still in clinical trial and completed the Letter six months before Jakafi became the first — and so far, the only — JAK2 inhibitor to be approved by US and European regulators. Since that time there has been extensive reporting of the Phase III COMFORT trials and substantial numbers of MF patients starting therapeutic Jakafi regimens.
The two central findings of this Letter come at a critical time for MF patients and caregivers. In cases where patients have terminated JAK2 inhibitor therapy because Jakafi was no longer effective, the finding of resensistization could be a new lease on life.
And in putting forth a possible reason in heterodimerization for the persistence of disease burden despite JAK2 inhibition, the work provides a platform for strategic assessment of new drug targets and therapeutic approaches.
And there’s one final conclusion
The JAK2 allele burden does not decline during extended inhibitor therapy.
Chronic treatment with inhibitor results in “stabilization of activated JAK2 facilitating the formation of heterodimers.” This persistent JAK2 activation despite formation of hetereodimers suggests blood production continues to be dependent on JAK2 and treatments that “lead to JAK2 degradation (HSP90, heat shock protein inhibitors or HDAC, histone deacetylace inhibitors) that retain the ability to inhibit JAK2 …have the potential to improve therapeutic efficacy in patients with MPN.“
The Mayo Clinic’s Dr. Ruben Mesa agrees. ” This is an important paper. Combination trials with HDAC inhibitors and JAK2 inhibitors are ongoing,” he says.
Take me back to the Contents
© Zhenya Senyak and MPNforum.com, 2012. Unauthorized use and/or duplication of this material without express and written permission from this blog’s author and/or owner is strictly prohibited. Excerpts and links may be used, provided that full and clear credit is given to Zhenya Senyak and MPNforum.com with appropriate and specific direction to the original content.