ALBERT

Description: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a subtype of high-risk B-precursor ALL (B-ALL) that carries a high risk of relapse after conventional chemotherapy (Mullighan et al, N Engl J Med. 2009). Rearrangements in CRLF2, leading to overexpression of the receptor for the cytokine thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALLs and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010; Tasian et al, Blood 2014). Previous studies established that combining a tyrosine kinase inhibitor (TKI) with an mTOR inhibitor provides greater anti-leukemia efficacy than a TKI alone in Ph+ B-ALL (Janes et al, Nat. Med. 2013). While allosteric mTOR inhibitors such as rapamycin only partially block mTORC1 and do not directly inhibit mTORC2, second-generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) efficiently block both mTOR outputs and show greater efficacy when combined with TKIs. In this study, we investigated anti-leukemia efficacy and intracellular signaling networks in Ph-like CRLF2+ ALL models treated with combinations of a type I or type II JAK-2 inhibitor and a TOR-KI. The inhibitors were tested in human B-precursor Ph-like ALL cell lines MUTZ5 (IGH@-CRLF2 translocation, JAK2 R683G mutation) and MHH-CALL-4 (IGH@-CRLF2 translocation, JAK2 I682F mutation), B-ALL cell line REH (CRLF2wt), and primary CRLF2+ xenograft cells in vitro. For signaling and growth inhibition studies, cells were stimulated with 25 ng IL-7 or TSLP for 30 min, then with JAK2 type I inhibitor ruxolitinib (500nM) or type II inhibitor NVP-BBT594 (500nM) (Andraos et al., Cancer Discov. 2012) and allosteric mTOR inhibitor rapamycin or TOR-KI AZD2014. Effects on intracellular signaling were determined by phospho-flow cytometry. Anti-leukemia effects were characterized by viable cell counts and annexin V flow cytometry. In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (p-JAK2(Tyr1008), p-STAT5(Ty694)) and AKT/pS6 signaling (p-AKT(Ser473), p-rS6(S235/236) (Fig. 1A). Stimulation with IL-7, mimicking support by the normal bone marrow environment, induced a lesser degree of activation of these phospho-proteins, except for p-4EBP1(T37/46), which was constitutively highly expressed in these cells and further induced by IL-7. These findings warranted combination studies of JAK2 and mTOR inhibitors. JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, and S6 activation, yet failed to decrease p-4EBP1 (Fig. 1A). AZD2014 but not rapamycin fully inhibited p-4EBP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth arrest in CRLF2+ cells (Fig. 1A, C). In turn, combination of ruxolitinib and AZD2014 further reduced cell proliferation but did not induce apoptotic cell death (Fig. 1B, D). Recent studies indicate persistence of JAK2-mutated cells in myeloproliferative neoplasms upon long-term exposure to a type I JAK2 inhibitor, mediated by JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2, in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of STAT5 phosphorylation; in contrast, BBT594 diminished both p-JAK2 and p-STAT5. Unexpectedly, BBT594 induced apoptotic cell death in both MUTZ5, MHH-CALL-4 (Fig 1B) and in ALL blasts recovered from primary CRLF2+ xenograft and grown in OP9 in vitro co-culture; the combination of BBT594 with AZD2014 increased apoptosis and reduced cell viability even further, in both cell lines and in stroma-attached primary ALL cells. In summary, these results suggest that efficient blockade of JAK2/STAT5 with a type II JAK2 inhibitor translates into cell death of JAK2-addicted CRLF2-rearranged cells and may have the capacity to eliminate JAK2-mutated clones. Concomitant blockade of TORC1 signaling with a TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4EBP1 and causes synthetic lethality, providing avenues for novel, rationally designed combinatorial regimens in this subset of Ph-like B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: GNB1 encodes a beta subunit (Gβ) of heterotrimeric G proteins, which mediate signals downstream of G protein coupled receptors (GPCRs). We isolated a somatic mutant of GNB1 (K89E) by functional screening of a cDNA library derived from a blastic plasmacytoid dendritic cell neoplasm (BPDCN). A search of cancer genome databases identified recurrent mutations in GNB1 and the highly related protein GNB2. GNB1/2 K89E/T were found in B cell acute lymphoblastic leukemia (B-ALL) (1 case), follicular lymphoma (1) and myelodysplastic syndrome (MDS) (1) as well as BPDCN (1). Interestingly GNB1 K57E/T mutations were found only in myeloid diseases: [acute myeloid leukemia (2), atypical CML (2), polycythemia vera (1) and MDS (6)], while GNB1 I80N/T were found predominantly in B cell diseases [CLL (2), FL (2), DLBCL (1) and MDS (1)]. These mutated codons are all located on the GNB1 protein surface that is critical for interactions between Gβ and alpha subunits (Gα) or downstream effectors. Immunoprecipitation followed by mass spectrometry demonstrated that GNB1 K57E, I80T and K89E mutants failed to bind Gα, including GNAI2/3, GNA11/Q and GNA13 that are normally bound by wild-type (WT) GNB1. All mutations affecting these codons promoted cytokine-independent growth of human TF1 myeloid cells or mouse BaF3 lymphoid cells with activation of MEK/ERK and mTOR/PI3K pathways. Pertussis toxin treatment did not affect GNB1-dependent ERK activation or cell growth, implying a Gα-independent pathway. To investigate the function of GNB1 mutations in vivo, we performed a mouse bone marrow transplantation (BMT) experiment using wild-type and Cdkn2a-deficient donors. Loss of the cell cycle regulator CDKN2A is common in BPDCN, B-ALL, and several other hematologic malignancies. Bone marrow cells were isolated from 5-FU treated donor mice and infected with retrovirus expressing GNB1 WT, K57E, I80T or K89E. Transplantation of GNB1 mutant-expressing Cdkn2a-deficient bone marrow resulted in myeloid dendritic cell neoplasms that were CD11b+, CD11c+, CD19-, B220-, and CD3-. GNB1 mutants did not induce tumors in WT bone marrow after 12 months of observation suggesting that GNB1 requires additional cooperating mutations such as Cdkn2a loss. We performed the same BMT experiment using Cdkn2a-deficient bone morrow cells without 5-FU pretreatment. We found thatGNB1 I80T and K89E mutants induced a progenitor B cell ALL (CD11b-, CD11c-, CD19+, CD3-, TdT+). These data suggest that GNB1 mutations can promote tumorigenesis in more than one cell lineage, as observed in patients. In vivo treatment of the myeloid neoplasm with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, we noted that GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with the mutant kinases resulted in relative resistance to treatment with the corresponding kinase inhibitor in each context. Thus, GNB1 and GNB2 mutations confer transformation and targeted therapy resistance across a range of human tumors and may be targetable with inhibitors of PI3K/mTOR signaling. Disclosures Gotlib: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel Support Other. Deininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy. Tyner:Constellation Pharmaceuticals: Research Funding.

Description: Approximately 10% of B-ALLs harbor CRLF2 rearrangements, which may portend a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity in vitro or in vivo (Weigert et al. J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al. Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation, which may overcome this resistance. When assayed in MHH-CALL4 cells harboring a CRLF2/IGH rearrangement and JAK2 I682F mutation, the type II JAK2 inhibitors NVP-BBT594 and NVP-CHZ868 were 10-35-fold more potent than the type I JAK2 inhibitors NVP-BSK805 and NVP-BVB808. Similarly, in Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the IC50 for CHZ868 was 5-20-fold lower than the IC50s for BSK805 and BVB808. Unlike type I inhibitors, which induce paradoxical hyperphosphorylation of JAK2, CHZ868 completely blocks JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not alter sensitivity to CHZ868. CHZ868 abrogates STAT5 phosphorylation in Ba/F3 cells expressing CRLF2 with JAK2 R683G/Y931C while BVB808 does not. CHZ868 is the first type II JAK2 inhibitor amenable to in vivo use. We assessed its efficacy in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/- or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Splenocytes from patient-derived xenografts (PDXs) treated with CHZ868 in vivo for 3 days are more primed for apoptosis as demonstrated by a 2-6-fold EC50 reduction for PUMA permeabilizing activity compared to vehicle. Transcriptional profiling of splenocytes from CHZ868-treated PDXs revealed downregulation of critical survival pathways including E2F1, STAT3, and AKT-mediated signaling. Of note, 2 of the most downregulated genes are STAT targets, PIM1 and Myc. Mice treated for 5-6 days with CHZ868 had significant reductions in spleen size and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p30 clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P displayed cross-resistance to CHZ868, while sensitivity to type I inhibitors was not affected. Structural modeling of the JAK2 JH1 domain suggested that L884P alters the binding pocket for type II inhibitors. JAK2 L884P is homologous to an EGFR L747P activating mutation, which destabilizes the P-loop and C-helix portion of the kinase domain (He et al. Clin Cancer Res 2012). The fact that L884P was reported in two B-ALL patients lacking additional JAK2 mutations (Torra et al. Blood (ASH Annual Meeting Abstracts) 2010) raised the possibility it was also an activating mutation. We confirmed L884P is an activating allele, as Ba/F3 cells expressing CRLF2, IL7R, and JAK2 L884P proliferated in the absence of TSLP ligand. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents currently used in B-ALL treatment. Dexamethasone was the most highly synergistic with CHZ868 in MHH-CALL4 cells. To assess the combination in vivo, we treated mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL with vehicle, CHZ868, dexamethasone, or CHZ868 + dexamethasone for 14 days post engraftment. CHZ868 treatment prolonged survival compared to vehicle (p

Description: Background and rationale: Philadelphia chromosome-like acute lymphoblastic leukemia ("Ph-like ALL") is a subtype of high-risk B-precursor ALL (B-ALL), which carries a high risk of relapse with conventional chemotherapy(Roberts et al, N Engl J Med. 2014). Rearrangements in CRLF2, leading to overexpression of cytokine receptor for thymic stromal lymphopoietin (TSLP), are present in approximately 50% of Ph-like ALL and are associated with hyperactive JAK/STAT and PI3K/mTOR signaling (Harvey et al, Blood 2010;Tasian et al, Blood 2014).In addition,JAK2 fusion proteins, such as PAX5-JAK2 represent a novel class of JAK2-driven cellular transformation in B-ALL (Dagmar et al, Blood 2015). Our prior studies in Ph+ B-ALL established that combining tyrosine kinase inhibitors (TKIs) with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs) provides greater anti-leukemia efficacy compared to TKIs in Ph+ ALL (Janeset al, Nat. Med. 2013). In this study, we investigated anti-leukemia efficacy and intracellular signaling networks upon combination of type I or type II JAK2 inhibitors and TOR-KIs in JAK2-driven Ph-like ALL models. Methods. The human B-precursor Ph-like ALL cell lines MUTZ5 (which harborsIGH-CRLF2 translocation and JAK2 R683G mutation), MHH-CALL-4 (IGH-CRLF2 translocation and JAK2 I682F),Reh (ETV6-RUNX1 B-precursor ALL cell line)and mouse Arf-null PAX5-JAK2-MIG + IK6-MIR(IL7-dependent primary Arf-/- pre-B cells expressing the dominant negative Ikaros isoform IK6 with PAX5-JAK2 fusion protein) were studied. Signal transduction inhibitors (STIs): JAK2 type I inhibitor ruxolitinib and type II inhibitor NVP-BBT594 (Andraos et al., Cancer Discovery 2012); allosteric mTOR inhibitor rapamycin or mTOR-KI AZD2014. Effects on intracellular signaling were determined using phospho-flow cytometry and Westernblot analysis. Anti-leukemia effects were quantified using CellTiter-Glo viability assay and annexin V flow cytometry. Results. In vitro stimulation of CRLF2-rearranged cells with TSLP robustly induced JAK/STAT signaling (Fig 1D). JAK2 inhibition with ruxolitinib or BBT594 efficiently inhibited TLSP-induced STAT5, AKT, ERK and S6 activation, yet failed to affect4E-BP1 activation. The TOR-KI AZD2014 but not rapamycin fully inhibited phosphorylation of 4E-BP1, consistent with efficient inhibition of TORC1, and caused profound cell cycle arrest and growth inhibition of Ph-like cells. Combination of ruxolitinib and AZD2014 further inhibited cell proliferation, yet did not induce apoptotic cell death. Recent studies indicate persistence of JAK2-mutated cells upon chronic exposure to type I JAK2 inhibitors, through an adaptive resistance mechanism involving JAK2 heterodimerization and reactivation of JAK-STAT signaling (Koppikar et al., Nature 2012). We therefore compared the in vitro efficacy of ruxolitinib and BBT594, a type II JAK2 inhibitor that retains the ability to bind inactive JAK2 in Ph-like ALL cells. In MUTZ-5 but not in MHH-CALL-4 cells, ruxolitinib increased JAK2 activation loop phosphorylation (p-JAK2-Tyr1008) despite suppression of p-STAT5; in contrast, BBT594 diminished bothp-JAK2 and p-STAT5 in both cell lines. Unexpectedly, BBT594 induced apoptotic cell death in all JAK2-driven Ph-like ALL cell lines MUTZ5, MHH-CALL-4 and Arf-null PAX5-JAK2+IK6, but not in REH cells. Combination of BBT594 with AZD2014 further inhibited phosphorylation of JAK2, AKT, 4E-BP1 and eIF4E, and synergistically induced apoptosis and reduced cell viability in Ph-like ALL cell lines(combination index: MUTZ5, 0.71; MHH-CALL-4, 0.57; Arf-nullPAX5-JAK2+ IK6, 0.81). Of importance, BBT594 and AZD2014 combination induced apoptosis in five JAK2-mutant Ph-like ALL xenograft primary samples. In summary, these results suggest that efficient blockade of JAK2/STAT5 with type II JAK2 inhibitors translates into cell death of mutant JAK2-driven Ph-like ALL cells. Furthermore, concomitant blockade of TORC1 signaling with TOR-KI reduces B-ALL cell proliferation through potent inhibition of 4E-BP1 and causes synthetic activity, providing avenues for novel rationally designed combinatorial regimens in this subset of Ph-like B-ALL. The in vivo studies to test these hypotheses are ongoing using patient-derived xenografts. Disclosures Jabbour: Pfizer: Consultancy, Research Funding. Tasian:Incyte: Consultancy; Gilead: Research Funding. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Description: To identify new oncogene alleles directly from primary tumor specimens, we generate and screen cDNA libraries from patient samples for gain-of-function alterations that can substitute for cytokine signaling in cytokine-dependent cells. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia of plasmacytoid dendritic cells with a dismal prognosis. No driver oncogenes have been identified in cases of BPDCN. Screening of a cDNA library generated from a BPDCN resulted in multiple cytokine-independent clones that expressed a full-length transcript of GNB1 with a K89E mutation. GNB1 encodes a beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, including GNAQ/GNA11 in uveal melanoma and GNAS in pituitary tumors, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. ERK and AKT activation by GNB1 K89E were confirmed by western blotting. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex assay of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. To assay the transforming effects of GNB1 K89E in vivo, we transduced GNB1 (wild-type or K89E) into bone marrow from Cdkn2a-deficient donors after 5-FU treatment and transplanted into wild-type recipients. We opted to utilize Cdkn2a-deficient donors as the loss of CDKN2A is common in cases of BPDCN. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal malignancy characterized by pancytopenia and massive hepatosplenomegaly. Spleens were infiltrated by large, spindly cells with extensive dendritic projections, as well as extensive fibrosis that completely effaced the normal splenic architecture. The cells were negative for T-cell (CD2, CD3) and B-cell (CD19, B220) markers but positive for the dendritic cell/macrophage markers MAC-2 and MAC-3. Further characterization by flow cytometry demonstrated that the cells infiltrating the spleen were CD8, CD103, MHC class II, CD26, FLT3 and CD11c positive, consistent with neoplastic dendritic cells. Serial transplantation of splenic cells from five different GNB1 K89E-transplanted mice into secondary wild-type recipients resulted in 100% fatality within 50 days. We searched published datasets from exome, transcriptome and whole genome sequencing of hematologic malignancies for GNB1 mutations. We identified one case of K89E in B-cell acute lymphoblastic leukemia (ALL), four cases with I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, six cases with K57E/T in myeloid neoplasms, and D76G in T-cell ALL. Expression of any of these alleles but not wild-type GNB1 was sufficient to promote cytokine-independent growth of human TF1 cells. The published structure of GNB1 (Ford et al. Science 1998) reported a small number of residues, including K57, I80 and K89 that mediate interactions with both G-alpha subunits and effector proteins. In fact, affinity purification followed by MS using tagged GNB1 (wild-type, I80T and K89E) demonstrated that, unlike wild-type GNB1, the GNB1 mutants fail to bind distinct Gα subunits. The repertoire of protein interactors, which includes potential G protein effectors, also differed between different GNB1 alleles. Thus, gain-of-function mutations in GNB1 occur across a broad range of hematologic malignancies, modify essential interaction G-protein subunit interactions, can drive in vivo transformation, and activate targetable downstream kinases. Disclosures: Tyner: Incyte Corporation: Research Funding.