Adaptive Reactivation of Signaling Pathways As a Novel Mechanism of Resistance to JAK Inhibitors in Ph-like ALL
Authors: Hurtz Christian , Tasian Sarah K , Wertheim Gerald , Astles Rachel , Zebrowski Alexis , Perl Alexander E. , Carroll Martin 
Published: October 2019

Abstract
Background:
Ph-like ALL was first described in 2009 and was accepted as a novel B-ALL subgroup by the WHO in 2016. Ph-like ALL is characterized by different chromosomal rearrangements involving CRLF2, ABL1, JAK2, and EPOR. In addition, mutations in PAX5, IKZF1, and JAK2 are very common. The gene expression profile of this disease is very similar to Ph+ ALL which is characterized by the BCR-ABL1 oncogene. We have previously shown that Ph-like ALL is a high risk disease with a poor prognosis and constitutive activation of cytokine receptor signaling pathway (Tasian et al., submitted). Our current study is focusing on redundant signaling activation in Ph-like ALL that allows the cells to survive cytokine receptor inhibition.
Results:
We and others have shown that Ph-like ALL have constitutive activation of the cytokine receptor signaling pathway evident by high expression levels of pSTAT5. Xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with Ruxolitinib a JAK1/2 inhibitor. However, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. To understand these results, we have initially used Ph-like ALL cell lines. Ruxolitinib treatment of such cell lines decreases survival but does not eliminate all viable cells. In addition, Ruxolitinib leads to down regulation of pSTAT5, pSTAT3, pAKTT308 as well as pAKTS473. To further study the mechanism of resistance to JAK inhibition we performed a time course experiments and found that as early as 48-72 hours after the initial JAK inhibitor treatment Ph-like ALL cell are able to reactivate pSTAT5 and pAKT. This effect was not due to drug inactivation. Based on this observation, we hypothesized that adaptive signaling through PI3 kinase protects Ph-like ALL cells from cell death. Interestingly, PI3K inhibition (Idelalisib) alone had only a minor effect on cell viability and cell proliferation and the Ph-like ALL cells were able to reactivate the signaling network within 48-72h. Dual treatment with JAK1/2 and PI3 kinase inhibition significantly increased the apoptotic effect and stopped proliferation. In addition, the dual treatment induced a long lasting inhibitory effect with no signs of pAKT reactivation. Strikingly, combining JAK and PI3K inhibition with Methotrexate an antimetabolite that is used for the treatment of ALL patients significantly increased the apoptotic effect of the combinatory treatment. Overall, these results demonstrate that adaptive signaling with reactivation of the PI3K signaling cascade protects Ph-like ALL cells from inhibition of JAK kinases and suggests that dual kinase inhibitor therapy may increase response in the clinic. Experiments studying how reactivation occurs and the in vivo response to dual JAK-PI3K inhibition are in progress.
Clinical relevance:
Older patients (>40 years) often do not qualify for induction chemotherapy as they do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens that can improve their clinical outcome. While children with ALL currently have a 5-year survival rate of 90% adults especially older adults have a very poor clinical outcome with survival rates of about 40%. We therefore combined JAK1/2 and PI3K inhibitory treatments with methotrexate a frequently used antimetabolite that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far the first study using specific signaling inhibitors combined with an antimetabolite treatment in Ph-like ALL.
Conclusion:
These findings identify that Ph-like ALL are able to reestablish their signaling networks. Based on our finding it is important to target more than one signaling pathways to fully inhibit cell proliferation and cell survival. We propose to treat Ph-like ALL patients with a combinatory treatment of JAK1/2 and PI3K inhibitor and currently used antimetabolite drugs to reduce the risk of relapse and to improve the clinical outcome of patients with Ph-like ALL.

Disclosures
Perl: Seattle Genetics: Consultancy; Actinium Pharmaceuticals, Inc.: Research Funding.