ALBERT

Description: Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. The maintenance of a proper balance between HSC self-renewal and differentiation requires growth factors, cytokines, and chemokines, most of which activate the phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of the AKT pathway is frequently observed in tumors, making it a desirable target for cancer treatment. Since several PI3K inhibitors are now in clinical use, it is critical to determine the roles of PI3K in adult HSCs. However, the specific roles of PI3K in HSC function are poorly understood. Hematopoietic cells express three Class IA catalytic PI3K isoforms (P110α, β, and δ), which can all transduce growth factor and cytokine signals, and can compensate for one another in some cell types. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but are dispensable for HSC function. To uncover the potentially redundant roles of PI3K isoforms in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells using MX1-Cre, and germline deletion of p110δ. TKO mice develop pancytopenia, which is also observed upon transplantation of TKO bone marrow. Competitive repopulation assays reveal a defect in long-term multi-lineage chimerism. Surprisingly, loss of Class 1A PI3K causes significant expansion of donor-derived long-term (Lin-cKit+Flk2-CD150+CD48-) and short-term (Lin-cKit+Flk2-CD150-CD48-) HSCs in the bone marrow, but not committed progenitors. This phenotype could not be explained by alterations in HSC cell cycling or apoptosis in TKO HSCs. TKO transplant recipients also have dysplastic features in the bone marrow. Methylcellulose plating assays of TKO bone marrow revealed a relative increase in granulocyte erythroid macrophage megakaryocyte (GEMM) colonies and extended serial replating, suggesting increased self-renewal. Thus, our data are consistent with impaired HSC differentiation upon deletion of all Class IA PI3K isoforms, which leads to dysplastic changes. RNA sequencing of sorted long-term HSCs from the bone marrow of TKO transplant recipients revealed the enrichment of human and mouse HSC signatures, and the downregulation of DNA repair gene sets and RNA splicing gene sets in TKO HSCs. Interestingly, we also observed downregulation of autophagy gene sets in TKO HSCs. Macroautophagy has been shown to be essential for the maintenance of HSC metabolism and self-renewal. Analysis of the autophagosomal marker LC3-II in TKO HSCs revealed a decrease in autophagy upon growth factor deprivation. Surprisingly, we observed an increase in MTOR activation in TKO cKit+ bone marrow cells via compensatory signaling through the MAPK pathway. Given that MTOR is a known negative regulator of autophagy, this is consistent with the observed autophagy decrease in TKO HSCs. Additionally, we found that autophagy can still be induced in TKO HSCs with the MTOR inhibitor rapamycin. Furthermore, rapamycin treatment impairs serial replating of TKO bone marrow cells. In conclusion, we found that inactivation of all Class 1A PI3 kinases leads to impaired HSC differentiation, likely due to a defect in autophagy induction in response to growth factor deprivation. Disclosures No relevant conflicts of interest to declare.

Description: The phosphoinositide 3-kinase (PI3K)/AKT pathway is commonly dysregulated in human malignancies, including leukemia. AKT, a downstream effector of PI3K, is constitutively phosphorylated in myeloproliferative disease (MPD) and acute myeloid leukemia (AML) patient samples, suggesting that the PI3K/AKT pathway may be an attractive therapeutic target. In myeloid malignancies, this pathway is most commonly activated not by mutations in PI3K, AKT or loss of PTEN, but rather by mutations in a spectrum of upstream tyrosine kinases, such as BCR-ABL, ETV6-PDGFRb, FIPL1-PDGFRa, JAK2V617F, or FLT3-ITD. To further understand the contribution of PI3K/AKT activation to disease pathogenesis, we modeled the activation of AKT in myeloid neoplasms by such upstream effectors using a myristoylated allele of AKT (myr-AKT) that is constitutively activated. Bone marrow from 5-fluorouracil-primed C57 Bl/6 donor mice was transduced with a bicistronic retrovirus expressing myr-AKT and enhanced green fluorescent protein (EGFP) or control retrovirus expressing EGFP alone, and transplanted into 30 and 5 lethally irradiated syngeneic recipients, respectively. The myr-AKT transplant recipients had a median survival of 53 days. Of 30 myr-AKT mice, 27 (90%) developed a myeloproliferative disease (MPD), characterized by splenomegaly, hepatomegaly, expanded Mac1+Gr1+, Mac1+ckit+, and CD71+Ter119+ populations in the bone marrow and spleen, and increased splenocyte myeloid colony formation. Of these 27 myr-AKT mice with MPD, 19 (70%) also had thymic T cell lymphoma, characterized by infiltration of the thymus, heart, lungs, and muscle with CD4+/CD8+ lymphoblasts. Three of 30 (10%) myr-AKT mice developed acute myeloid leukemia (AML) with phenotypic attributes of erythroleukemia (AML M6) in humans, characterized by infiltration of the spleen, liver and bone marrow with CD71hickit+ blasts. Control EGFP recipients had no evidence of disease. Splenocytes from mice with AML and thymocytes from mice with T cell lymphoma caused disease when transplanted into secondary recipients, whereas splenocytes from mice with MPD were unable to transplant disease. Of note, we observed that myr-AKT expression caused impaired engraftment in recipient mice, as evidenced by a decrease in the %EGFP in the bone marrow over time. Although myr-AKT expressing cells can home normally to the bone marrow, myr-AKT significantly impairs the lodging ability of transduced bone marrow in irradiated recipients by 2 weeks after transplant. Furthermore, we observed an increased rate of apoptosis in myr-AKT-expressing bone marrow and spleen cells in myr-AKT recipient mice. Taken together, these data suggest that constitutive activation of AKT paradoxically increases apoptosis and impairs engraftment of transduced cells, but demonstrate that constitutive activation of AKT alone nonetheless recapitulates the spectrum of human myeloid neoplastic phenotypes associated with activation of upstream tyrosine kinase effectors.

Description: The PI3 kinase (PI3K) signaling pathway is activated by most hematopoietic growth factors and chemokines that orchestrate normal adult hematopoiesis. Hematopoietic cells express four different catalytic isoforms of PI3K (p110α, β, δ, and γ), each encoded by a different gene (pik3ca, pik3cb, pik3cd and pik3cg, respectively). The Class IA (p110 α, β, δ) and Class IB (p110γ) enzymes use different regulatory subunits and are activated by different sets of upstream signals. While pik3ca and pik3cb are expressed ubiquitously, pik3cd is enriched in leukocytes. The roles of PI3K in adult hematopoiesis have been difficult to investigate using gene targeting in mice, since two of the isoforms (p110α and p110β) are required during embryogenesis. However, mice with germline deletion of pik3cd are viable and fertile with normal blood counts (Jou et al, MCB, 2002. 22(24):8580-91). Using the Mx1-Cre system to conditionally delete pik3ca in adult hematopoietic stem cells (HSCs), we previously showed that p110α is dispensable for adult HSC function (Gritsman et al, J Clin Invest 2014;124(4):1794-1809). We now report that conditional deletion of pik3cb using Mx1-Credoes not affect blood counts, bone marrow cellularity, survival, bone marrow HSC or progenitor numbers, or hematopoietic reconstitution in the transplantation setting. Therefore, none of the individual Class IA PI3K isoforms have critical roles in HSCs. However, it is still unclear whether these isoforms can substitute for each other in adult HSCs, or whether they are truly dispensable for HSC function. To distinguish between these two possibilities and to uncover potentially redundant roles for PI3K isoforms in hematopoiesis, we have generated compound conditional knockout mice with the genotypes: pik3ca-lox/lox;pik3cd-/-;Mx1-Cre ("p110α-/-;p110δ-/-") and pik3ca-lox/lox;pikcb-lox/lox;Mx1-Cre ("p110α-/-;p110β-/-"). Interestingly, compound deletion of p110α and p110δ in HSCs results in significantly reduced white cell counts and anemia (Gritsman et al, Blood (ASH Annual Meeting Abstracts), Nov 2012; 120: 2322). In addition, p110α-/-;p110δ-/- mice have reduced numbers of bone marrow multipotential progenitors (MPPs), common myeloid progenitors (CMPs), and common lymphoid progenitors (CLPs), while HSC numbers are preserved. Furthermore, deletion of both p110α and p110δ leads to reduced B cell repopulation in competitive repopulation assays. This suggests that p110α and δ have redundant roles in HSC differentiation and B cell specification. In contrast, we found that compound deletion of p110α and p110β has no effect on long-term competitive repopulation of any lineages for up to 20 weeks. We also observed normal donor chimerism in the HSC, MPP, and progenitor populations in transplant recipients of p110α-/-;p110β-/- bone marrow. After non-competitive transplantation, recipients of p110α-/-;p110β-/- bone marrow maintained normal blood counts over time. This indicates that, while p110δ is sufficient to support hematopoietic reconstitution of all lineages in the transplantation setting, p110β cannot support normal HSC differentiation by itself, particularly into the B cell lineage. However, p110α-/-;p110δ-/- mice do not develop pancytopenia, and repopulation of the myeloid and T cell lineages is still largely preserved, suggesting that long-term HSC function is intact. To determine whether HSC self-renewal and myeloid reconstitution after transplantation are possible in the absence of all three Class IA PI3Ks, we have generated triple knockout mice with the genotype pik3ca-lox/lox;pikcb-lox/lox;pik3cd-/-;Mx1-Cre ("TKO"). Analysis of the hematopoietic phenotype of TKO mice will be presented. These results will be valuable in predicting future hematologic toxicity in ongoing clinical trials with isoform-selective PI3K inhibitors or pan-PI3K inhibitors. Disclosures No relevant conflicts of interest to declare.

Description: Key Points North American ATLL has a distinct genomic landscape with a high frequency of prognostic epigenetic mutations, including EP300 mutations. ATLL samples with mutated EP300 have compromised p53 function and are selectively sensitive to decitabine treatment.

Description: Key Points Targeting of PAK1 inhibits primary AML and MDS patients' cells including leukemia stem cells but spares healthy stem and progenitor cells. Inhibition of PAK1 induces differentiation and apoptosis of AML cells through downregulation of MYC and a core network of MYC target genes.

Description: Activation of MAPK signaling is characteristic of many cancers, and approximately 15% of AML patients carry activating RAS mutations. The PI3K pathway is also constitutively activated in AML. Both pathways interact with each other extensively and provide compensatory signaling when one pathway is inhibited. It has been demonstrated in vitro that concurrent inhibition of both pathways is effective in blocking the proliferation of AML cell lines through an immediate decrease in pAkt and pErk, leading to inhibition of pS6 (Gritsman et al, J Clin Invest, 2014, 124(4):1794-1809). The combination of alpelisib (BYL-719), which inhibits PI3K (p110alpha), and binimetinib (MEK-162), which inhibits MEK (MAPK pathway), was tested in various tumor types in the Phase Ib Clinical Trial CMEK162x2109. We present here the clinical data and correlative phosphoprotein analysis of 6 patients with relapsed refractory RAS-mutated AML treated at Massachusetts General Hospital on Extension Arm 4A of this trial. Entry to this trial was restricted to adult patients with RAS-mutated AML previously treated with 1 or 2 prior chemotherapy regimens or for whom there was no known effective therapy. Patients received 200mg QD of alpelisib and 45mg BID of binimetinib concurrently and continuously with adjustments for toxicities. Hematologic toxicity could not be determined in these patients with active AML causing myelosuppression. There were responses in blood and/or bone marrow (BM) in 3 patients, but none made partial response criteria by IWG guidelines, mainly due to platelet counts 50% reduction in a third patient. A rise in absolute neutrophil count was seen in 3 patients, 2 from below 100/ul to above 500/ul, and in 1 from 5,320/ul to 12,797/ul during the first month. BM partial responses were seen in 2 patients, with blast percentages dropping from 39% to 10% in 1 patient and from 12% to 5% in another. We analyzed the effects of this drug combination on signaling targets by collecting peripheral blood samples on day 1 pre-treatment and at 6 and 24 hours post-initiation of treatment. In 4 patients, we performed analysis of the phosphorylation of Akt, ribosomal protein S6, Erk, STAT5, and STAT3 on timed peripheral blood samples by immunoblotting. In all 4 cases at baseline, pAkt, pErk, and pS6 were detectable, while pSTAT5 and pSTAT3 levels were variable. In 3 of 4 cases, we observed a transient decrease in pAkt at 6 hours, but then a rebound at 24 hours. In 3 of 4 cases, we observed a lack of sustained pS6 inhibition. We observed sustained pErk inhibition at 24 hours in 2 cases. One patient who had blood blast clearance, improvement in ANC, and a drop in bone marrow blasts, showed strong inhibition of pAkt and pERK at 6 hours, although pS6 did not decrease. In another case we performed phospho-flow cytometry on timed whole blood samples. We observed an increase in pAkt, pS6, and pErk from baseline at 24 hours, both in CD34+38+blasts, and in the primitive CD34+38- cells. This second patient showed no clinical benefit from the treatment in terms of blood blast count, ANC, or platelets. We also performed immunohistochemistry for pS6, pErk, pAkt, pSTAT5, pSTAT3, p-eIF4E, and Caspase 3 on BM sections obtained at diagnosis and at one month and two months post-initiation of treatment, when available. These studies generally showed either persistence or an increase in the pS6 and p-eIF4E signals, both indicators of mTORC1 activity, in post-treatment BM samples. Levels of pAkt, pErk, pSTAT5 and pSTAT3 were highly variable in the post-treatment bone marrow samples. In conclusion, we demonstrated some initial target inhibition with the concurrent use of alpelisib and binimetinib in a subset of patients. However, inhibition of late downstream targets was not sufficiently sustained to achieve consistent clinical benefit in our patients with RAS-mutated AML. While the strategy of concurrent inhibition of various critical signaling pathways remains interesting, sustained inhibition of downstream signaling may require a different dosing schedule of the two drugs. Given the incomplete inhibition of mTORC1 targets pS6 and p-eIF4E in most cases, the addition of a third agent to inhibit pathways causing cross-activation downstream of mTORC1 may be required. Disclosures Weinstock: Novartis: Consultancy, Research Funding. Fathi:Bexalata: Other: Advisory Board participation; Merck: Other: Advisory Board participation; Celgene: Consultancy, Research Funding; Seattle Genetics: Consultancy, Other: Advisory Board participation, Research Funding; Agios Pharmaceuticals: Other: Advisory Board participation.

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.