Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its ...effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT–expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT–expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.
Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region ...on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apcmin allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apcmin mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apcmin bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apcmin mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).
The causes for malignant progression of disseminated tumors and the reasons recurrence rates differ in women with different breast cancer subtypes are unknown. Here, we report novel mechanisms of ...tumor plasticity that are mandated by microenvironmental factors and show that recurrence rates are not strictly due to cell-intrinsic properties. Specifically, outgrowth of the same population of incipient tumors is accelerated in mice with triple-negative breast cancer (TNBC) relative to those with luminal breast cancer. Systemic signals provided by overt TNBCs cause the formation of a tumor-supportive microenvironment enriched for EGF and insulin-like growth factor-I (IGF-I) at distant indolent tumor sites. Bioavailability of EGF and IGF-I enhances the expression of transcription factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Combinatorial therapy with EGF receptor and IGF-I receptor inhibitors prevents malignant progression. These results suggest that plasticity and recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for patients with TNBC.
We report a
Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem ...cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to
JAK2V617F-positive MPN.
► Preclinical murine model of human PV in which MPN is serially transplantable ► Distinct cell populations responsible for disease initiation and MPN phenotype ► No significant selective competitive advantage for
Jak2V617F-expressing HSCs ► Treatment with a JAK2 inhibitor did not eradicate MPN-initiating population
RNA-binding proteins of the Musashi (Msi) family are expressed in stem cell compartments and in aggressive tumors, but they have not yet been widely explored in the blood. Here we demonstrate that ...Msi2 is the predominant form expressed in hematopoietic stem cells (HSCs), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of human MSI2 in a mouse model increases HSC cell cycle progression and cooperates with the chronic myeloid leukemia-associated BCR-ABL1 oncoprotein to induce an aggressive leukemia. MSI2 is overexpressed in human myeloid leukemia cell lines, and its depletion leads to decreased proliferation and increased apoptosis. Expression levels in human myeloid leukemia directly correlate with decreased survival in patients with the disease, thereby defining MSI2 expression as a new prognostic marker and as a new target for therapy in acute myeloid leukemia (AML).
The genes encoding RAS family members are frequently mutated in juvenile myelomonocytic leukemia (JMML) and acute myeloid leukemia (AML). RAS proteins are difficult to target pharmacologically; ...therefore, targeting the downstream PI3K and RAF/MEK/ERK pathways represents a promising approach to treat RAS-addicted tumors. The p110α isoform of PI3K (encoded by Pik3ca) is an essential effector of oncogenic KRAS in murine lung tumors, but it is unknown whether p110α contributes to leukemia. To specifically examine the role of p110α in murine hematopoiesis and in leukemia, we conditionally deleted p110α in HSCs using the Cre-loxP system. Postnatal deletion of p110α resulted in mild anemia without affecting HSC self-renewal; however, deletion of p110α in mice with KRASG12D-associated JMML markedly delayed their death. Furthermore, the p110α-selective inhibitor BYL719 inhibited growth factor-independent KRASG12D BM colony formation and sensitized cells to a low dose of the MEK inhibitor MEK162. Furthermore, combined inhibition of p110α and MEK effectively reduced proliferation of RAS-mutated AML cell lines and disease in an AML murine xenograft model. Together, our data indicate that RAS-mutated myeloid leukemias are dependent on the PI3K isoform p110α, and combined pharmacologic inhibition of p110α and MEK could be an effective therapeutic strategy for JMML and AML.
Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region ...on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apc(min) allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apc(min) mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apc(min) bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apc(min) mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).
The genes encoding RAS family members are frequently mutated in juvenile myelomonocytic leukemia (JMML) and acute myeloid leukemia (AML). RAS proteins are difficult to target pharmacologically; ...therefore, targeting the downstream PI3K and RAF/MEK/eRk pathways represents a promising approach to treat RAS-addicted tumors. The p110alpha isoform of PI3K (encoded by Pik3ca) is an essential effector of oncogenic KRAS in murine lung tumors, but it is unknown whether p110alpha contributes to leukemia. To specifically examine the role of p110alpha in murine hematopoiesis and in leukemia, we conditionally deleted p110alpha in HSCs using the Cre-loxP system. Postnatal deletion of p110alpha resulted in mild anemia without affecting HSC self-renewal; however, deletion of p110alpha in mice with KRAS.sup.G12D-associated JMML markedly delayed their death. Furthermore, the p110alpha- selective inhibitor BYL719 inhibited growth factor-independent KRAS.sup.G12D BM colony formation and sensitized cells to a low dose of the MEK inhibitor MEK162. Furthermore, combined inhibition of p110alpha and MEK effectively reduced proliferation of RAS-mutated AML cell lines and disease in an AML murine xenograft model. Together, our data indicate that RAS-mutated myeloid leukemias are dependent on the PI3K isoform p110alpha, and combined pharmacologic inhibition of p110alpha and MEK could be an effective therapeutic strategy for JMML and AML.
Abstract 3620
Poster Board III-556
PIK3CA, which encodes the p110α catalytic isoform of PI3 kinase (PI3K), is mutated in many human cancers, and is an attractive therapeutic target. However, PI3K may ...also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors which control hematopoietic stem cell (HSC) and progenitor proliferation, differentiation, and self-renewal, such as erythropoietin receptor (epoR), c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (α, β, δ). However, the roles of PI3K and its specific catalytic isoforms in normal HSC function are poorly understood. We hypothesized that signaling through the p110α isoform is important for hematopoiesis and HSC self-renewal. We have used the Cre-loxP system to delete p110α in the HSCs of adult mice by breeding p110αF/F mice to Mx1-Cre transgenic mice. p110αF/F;Mx1-Cre+ (Cre+) mice and their p110αF/F (Cre-) littermates were injected with PolyI-PolyC (pIpC) at 4-6 weeks of age to induce Cre-mediated excision at the PIK3CA locus specifically in hematopoietic cells. Deletion of p110α in the bone marrow (BM) was verified by PCR and by immunoblot. We observed that, by four weeks after pIpC treatment, Cre+ mice developed microcytic anemia compared with Cre- littermates, characterized by a decreased mean hemoglobin (p<0.0001) and decreased mean corpuscular volume, while white blood cell counts and platelet counts were unaffected. Cre+ mice also had significantly decreased spleen, liver, and thymus weights. Flow cytometry analyses of bone marrow and spleen cells revealed a relative block in erythropoiesis in the spleens of Cre+ mice, with expansion of the basophilic erythroblast population, and a decrease in the most mature nucleated erythroblast population. Colony assays of splenocytes in erythropoietin-containing methylcellulose medium revealed a 52% decrease in BFU-E colony formation by p110α-deleted cells in response to erythropoietin, suggesting that loss of p110α may lead to blunted epoR signaling (p=0.009). Multiparameter flow cytometry revealed that the overall number of Lin-Sca1+ckit+ (LSK) cells, which contains the HSC population, was increased two-fold in the BM of Cre+ mice compared with Cre- littermates (p=0.01). To determine whether loss of p110α affects long-term HSC self-renewal in vivo, we performed competitive repopulation assays, in which CD45.2+ BM cells from PIPC-treated Cre+ mice or Cre- controls were transplanted together with CD45.1+CD45.2+ competitor BM cells into lethally irradiated CD45.1+ recipient mice. Donor BM chimerism (%CD45.2+ cells) at 16 weeks was mildly reduced in the absence of p110α, but Cre+ cells were still capable of long-term reconstitution. In summary, we have found that the p110α catalytic isoform is specifically required for erythropoiesis, but has only a small role in HSC homeostasis and in differentiation of the other hematopoietic lineages. This suggests that pharmacologic targeting of p110α in cancer therapy may result in mild anemia, but should otherwise have minimal hematologic toxicity.
Gilliland:Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals, Inc.: Consultancy. Zhao:Novartis Pharmaceuticals, Inc.: Consultancy.
Abstract 1025
Poster Board I-47
Acute myeloid leukemia (AML) initiating cells reside within and utilize the bone marrow microenvironment, as a sanctuary to evade chemotherapy and to maintain ...self-renewal. Following treatment, these leukemia stem cells (LSC) re-emerge and reconstitute disease, leading to relapse. The canonical Wnt signaling pathway is frequently dysregulated in LSC and recent data indicates that Dkk1 (a potent endogenous Wnt inhibitor) may have a therapeutic role in treating AML. Microenvironment specific Dkk1 expression inhibits hematopoietic stem cell (HSC) Wnt and extinguishes HSC self-renewal in vivo, identifying the Wnt pathway as essential in normal HSC-niche homeostasis. We investigated the importance of bone marrow microenvironment Wnt signaling in LSC survival.
AML was generated using retroviral transduction of murine bone marrow with the MLL-AF9 fusion oncogene. We then assessed the potential for niche-directed Wnt inhibition of LSC using 2.3kbColl1alpha-Dkk1 transgenic mice in which Dkk1 expression is restricted to osteoblasts. AML was observed in the Dkk1 or wild type mice with similar disease latency and phenotype. AML was also observed in secondary transplant recipients, although there was a reduction of LSC (linlowcKithighSca-1-FcGRII/III+CD34+) derived from Dkk1 mice (LSC frequency 2.8% WT vs 1.6% Dkk1, p<0.05), correlating with a subtle prolongation in disease latency (n=15, 20 days WT vs. 24 days Dkk1, p<0.001).
To determine the status of Wnt signaling in MLL-AF9 AML, we generated AML in bone marrow derived from TOPGal reporter mice that harbor a Tcf/Lef responsive promoter with a LacZ reporter, and quantified LacZ expression or galactosidase protein levels. Wnt activation was increased following transformation of bone marrow with MLL-AF9 (relative TOPGal expression 1.35 empty vector vs 2.58 MLLAF9, p=0.03). To assess the effects of osteoblast-restricted Dkk1 expression in vivo, Wnt signaling was measured in LSC purified by high-speed multiparameter flow cytometry. Reporter activity (fluorescein di-β-D-galactopyranoside (FDG), Invitrogen) was unchanged in LSC from WT or Dkk1 recipients (Median fluorescent intensity 552 vs 542, p=0.85), indicating that, in contrast with normal HSC, Wnt signaling in LSC is relatively resistant to Dkk1 expression in the niche.
To better understand the mechanism of LSC resistance to Dkk1, we examined the homing and micro-localization of LSC in vivo using live, 3 dimensional two photon-confocal hybrid imaging of the bone marrow microenvironment. LSC proliferate with similar kinetics in Dkk1 or WT recipients (proliferating fraction 57.7% WT vs 50.3% Dkk1 LSC p=0.48). However, when compared to HSC, LSC home with less affinity to osteoblasts and may escape the effects of osteoblast specified Dkk1 expression through residence in a niche that is physically distant from endosteum (Median distance to osteoblast 18um WT vs 20.6um Dkk1 LSC, p=0.13).
Taken together, these data indicate that MLL-AF9 LSC can escape the normal HSC-niche homeostatic constraints regulated by Wnt, an observation that may have important therapeutic implications.
Scadden:Fate Therapeutics: Consultancy. Gilliland:Merck: Employment.
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