Oncogene-Independent Adaptation of Pre-B Cell Receptor Signaling Confers Drug Resistance and Signaling Plasticity in Ph-like ALL
Authors: Hurtz Christian , Wertheim Gerald , Loftus Joseph Patrick , Blumenthal Daniel , Lehman Anne , Li Yong , Manning Bryan , Cummins Katherine D , Burkhardt Janis K , Perl Alexander E. , Carroll Martin , Tasian Sarah K. 
Published: February 2020

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype occurring in 15-40% of older children, adolescents, and adults with B-ALL and is associated with high relapse rates and poor survival. Approximately 50% of Ph-like ALL cases have CRLF2 (cytokine receptor-like factor 2 rearrangements (CRLF2-R), often with concomitant JAK2 point mutations. We and others have shown that targeted inhibition of JAK/STAT and PI3K/AKT signaling in CRLF2-rearranged Ph-like ALL only partially inhibits leukemia proliferation. Our preliminary data indicates that modified B cell receptor signaling results in ERK activation and Ph-like ALL cell growth that appears independent of CRLF2-activated signaling. This study aimed to define the extent to which BCR signaling mediates drug resistance in Ph-like ALL after cytokine receptor signaling inhibition and to develop novel combinatorial treatment strategies.
Methods and Results: We first validated high basal levels of JAK/STAT and PI3K pathway phosphoproteins via immunoblotting of cell lysates from &gt;20 primary childhood and adult Ph-like ALL samples and patient-derived xenograft (PDX) models. Despite lack of B-cell receptor (BCR) μ heavy chain on Ph-like ALL cells by flow cytometry analysis, we detected high expression levels of the BCR-associated proteins CD79A, CD79B, and BLNK. We further identified constitutive phosphorylation of downstream BCR-associated signaling molecules, including SRC family kinases (SFKs), BTK, and ERK. Given these new observations, we then sought to delineate more precisely alternative pathways in Ph-like ALL (beyond JAK/STAT and PI3K) that may contribute to adaptive signaling regulation and confer resistance to kinase inhibitor monotherapy.
We hypothesized that JAK inhibitor (JAKi) resistance is mediated by compensatory activation of PI3K/AKT/mTOR through 'BCR-like' signaling via CD79/CD19/SRC/BTK/ERK. To test this hypothesis, we treated CRLF2-R Ph-like ALL cells in vitro with selective inhibitors of JAK1/2 (ruxolitinib) or PI3Kd (INCB050465) and assessed effects upon signaling via immunoblotting. We observed i) effective dephosphorylation of STAT5 and moderate dephosphorylation of AKT with full reactivation within 72h following JAKi or PI3Kdi monotherapy, respectively, ii) combined JAKi and PI3Kdi prevented PI3K pathway, reactivation, and iii) JAKi and PI3Kdi had no effects upon ERK signaling. These results suggested that oncogene-independent activation of BCR and PI3K pathway signaling occurs in Ph-like ALL. We then performed immunofluorescence analysis and detected distinct clustering and homodimerization of CD79B at the cell membrane of Ph-like ALL cells, as well as physical proximity of CD79B and CD19, suggesting that CD19 could be phosphorylated by SRC/SFKs independently of an activated BCR. Interestingly however, CRISPR-mediated deletion of CD19 or CD79B resulted in hyperactivation of ERK signaling. Finally, we tested if triple inhibition of JAK/STAT, PI3K, and SRC/SFKs signaling is sufficient to eradicate Ph-like ALL. Strikingly, combined JAKi (ruxolitinib), PI3Kdi (INCB050465), and SRCi (dasatinib) silenced STAT5, PI3K, and ERK signaling and induced near-complete cytotoxicity in vitro and in vivo in Ph-like ALL cell lines and patient-derived xenograft (PDX) models. While triple inhibitor treatment appeared well-tolerated in mice, we also assessed dual kinase inhibition of JAK2 and PI3K with dexamethasone (a known repressor of BCR signaling) as a more clinically relevant combinatorial strategy. We observed similarly robust inhibition of signaling pathways and leukemia eradication in vitro and in vivo in our Ph-like ALL cell lines and PDX models.
Conclusions: Children and adults with Ph-like ALL have a &gt;60% risk of relapse despite maximally intensive chemotherapy, demonstrating need for alternative therapeutic approaches. While earlier studies by our group and others have reported partial anti-leukemia activity of JAKi and PI3Ki monotherapies, our new data provide compelling evidence that three essential cooperating signaling pathways are required for Ph-like leukemogenesis. We postulate that triple JAK, PI3K, and SRC inhibition or dual kinase inhibition with chemotherapy can overcome innate compensatory mechanisms of signaling resistance and may in fact be necessary to eradicate Ph-like ALL in patients.

Disclosures
Perl: Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding; Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Bayer: Research Funding; BioMed Valley Discoveries: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy. Tasian:Gilead Sciences: Research Funding; Incyte Corportation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees.