Alice Shih

GLI2 inhibition abrogates human leukemia stem cell dormancy.

BackgroundDormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.MethodsTo test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.ResultsNotably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.ConclusionIn summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

Abstract 5217: RNA editing enzyme ADAR1 drives leukemia stem cell differentiation and self-renewal in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is the first cancer that was shown to originate from a genetic abnormality - the Philadelphia chromosome translocation, and production of its constitutively active protein tyrosine kinase product, BCR-ABL. The disease progresses slowly from chronic phase to accelerated phase, and later transforms to blast crisis (BC) stage. Cancer stem cells (CSCs) are a subset of tumor cells that have acquired certain treatment-resistant stem cell properties. High levels of RNA editing are associated with a primitive transcriptional program typical of human embryonic stem cells, and RNA editing plays an important role in both embryonic hematopoietic cell fate determination and in maintenance of normal hematopoiesis. Human RNA editing occurs primarily in secondary structures created by Alu retroelements and is carried out by enzymes such as the adenosine deaminase acting on RNA (ADAR) family. Among these, ADAR1 was also recently shown to be required for normal hematopoiesis by suppressing interferon-induced apoptosis. Our research focuses on dissecting the role of ADAR-mediated RNA editing in normal human hematopoietic progenitor cell development compared with malignant editing programs that may be activated in leukemia stem cells (LSC) during the progression of human CML. Our data demonstrates that BC LSC harbor increased levels of the interferon-responsive ADAR1 p150 isoform compared with chronic phase (CP) progenitors and normal cord blood progenitors. Expression of this isoform also exhibits a positive correlation with BCR-ABL expression levels - an effect which is specific to BC progenitors, suggesting that ADAR1 expression correlates with disease progression from CP to BC. In vitro hematopoietic progenitor assays with normal cord blood progenitors and CP samples transduced with lentiviral vectors overexpressing human ADAR1 reveals a significant shift in cell differentiation fate towards granulocyte-macrophage progenitor (GMP) colonies, which has been shown to be the initiating LSC population in CML Correspondingly, a progression towards erythroid lineage was observed in BC CML LSC transduced with lentiviral vectors expressing shRNA targeting ADAR1. Further qRT-PCR analyses revealed that the mechanism through which ADAR1 drives LSC and HSC differentiation towards myelopoiesis involves regulation of PU.1, which in turn inhibits GATA1 expression. Moreover, in vivo studies in a robust humanized CML mouse model showed a significant decrease in LSC serial transplantation potential of lentiviral shADAR1-transduced BC progenitors transplanted into neonatal RAG2-/-γc-/- mice. Together, these data support a crucial role for ADAR1 in cell fate determination and self-renewal potential of hematopoietic stem cells in both normal human progenitors and in malignant LSC that drive disease progression and therapeutic resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5217. doi:1538-7445.AM2012-5217

Abstract 974: A selective Notch1 mAb targets leukemia progenitor cells in T-ALL

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Difficulties in maintaining primary cultures of leukemia cells hampered efforts to investigate the biology of human T cell acute lymphoblastic leukemia (T-ALL) underscoring the need for a direct transplantation model to characterize leukemia progenitor cells (LPC) in vivo and as a paradigm for screening candidate drugs that inhibit self-renewal pathways active in T-ALL. Approximately, 50% of patients with T-ALL harbor NOTCH1 activating mutations that promote therapeutic resistance providing the impetus for developing selective NOTCH1-inhibitory therapeutic strategies. To investigate 1) whether a select subclone of T-ALL cells harbor a greater capacity to propagate disease in vivo than other clones, 2) to establish a humanized T-ALL LPC mouse model and 3) to test whether a selective NOTCH1-NRR/Fc (hN1) mAb inhibits LPC survival and self-renewal. Experimental Procedures: To facilitate non-invasive in vivo monitoring of leukemic engraftment, lentiviral luciferase transduced LPC were intrahepatically transplanted into neonatal immune deficient mice to establish humanized T-ALL LPC mouse models. These models were treated with hN1 mAb or a control IgG1 mAb at the dose of 10 mg/kg every 4 days for 21 days, and another group was treated with mouse IgG1 isotype control at the same dosing plan. Mice were sacrificed one day after the last dose. Thymus, spleen, liver and bone marrow (BM) were collected and analyzed by FACS. Some BM were sectioned for CD45, NOTCH1 and active Caspase 3 examination by immunohistochemistry. Results: Human CD34+ enriched cells maintained leukemic engraftment while an equivalent number of Lin+ cells did not. T-ALL CD34+ progenitors from 32 T-ALL LPC models established with NOTCH1 mutated T-ALL have a significant higher engraftment in BM when compared with those from 14 T-ALL LPC models established with Non-NOTCH1 mutated T-ALL. Human CD45+CD34+CD2+ population in serial transplant recipients was more prominent in NOTCH1 mutated samples. Human CD34+ populations were significantly reduced in both BM (p < 0.01, Student t test) and spleen (p < 0.05, Student t test) when the T-ALL LPC treated with hN1 mAb, while CD45+ populations were also significantly reduced in both BM and spleen (p < 0.05, Student t test). NOTCH1 expression level in human CD34+ cells in NOTCH1 mutated T-ALL was markedly reduced after hN1 mAb treatment when compared with IgG1 mAb treatment. NOTCH1 and CD45 positive cells were significantly reduced, while apoptosis was remarkably increased in the BM treated with therapeutic hN1 mAb when compared with those treated with IgG1 mAb. Intracellular domain of Notch1 was significantly reduced in the BM treated with hN1 mAb when compared with those treated with IgG1 mAb. Conclusions: 1. Human T-ALL LPC have enhanced NOTCH1 expression. 2. Human self-renewing T-ALL LPC are enriched in the CD45+CD34+CD2+ population. 3. A selective hN1 mAb inhibits human T-ALL LPC survival and self-renewal in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 974. doi:10.1158/1538-7445.AM2011-974

Abstract 4313: Mouse model of human T-cell acute lymphoblastic leukemia stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC T-cell acute lymphoblastic leukemia (T-ALL) is a very common malignancy diagnosed in children, accounting for 15% of all pediatric ALL cases. In pediatric T-ALL, 50% of patients harbor a Notch1 activating mutation1. Patients with relapsed T-ALL have a poor prognosis and thus it is important to understand the molecular mechanisms. Leukemia stem cells (LSC) play a key role in cancer propagation and have the capacity to self-renew and differentiate. LSC have been reported in T-ALL where, following in vitro culture, CD34+/CD4—— and CD34+/CD7——subfractions of T-ALL marrow were enriched for LSC capable of engrafting leukemia in NOD/SCID mice2. However, difficulties in maintaining primary cultures of leukemia cells have hampered investigations into the biology of T-ALL underscoring the need for a direct xenotransplant model for screening candidate drugs that inhibit self-renew pathways. Candidate LSC from T-ALL patient samples (n=10) were sorted using a FACSAria. Notch1, Hes1 and c-myc expression were analyzed in sorted cells by Q-PCR and Notch1 levels were measured by FACS. In addition, key genes were sequenced from some of the samples. To develop a mouse model for human T-ALL, sorted candidate LSC were lentivirally transduced with GFP-Luciferase fusion protein (GLF) and transplanted intrahepatically into neonatal T, B, and NK cell deficient mice3. Leukemic engraftment was monitored by in vivo bioluminescence imaging. The mice were sacrificed 8 weeks after transplant; hematopoietic organs were collected for FACS analysis of human CD2, CD7, CD34 and CD45 engraftment. Finally, to assay LSC self-renewal, engrafted human CD34+ cells from the bone marrow or thymus were transplanted into secondary and tertiary recipients. While Q-PCR and FACS data showed that Notch1 levels varied among different T-ALL patients, Notch 1 expression correlated with level of engraftment. We transplanted 10 T-ALL patient samples with higher Notch1 expression and 9 of 10 samples engrafted immunocompromised mice. Transplanted LSC could be tracked 4 weeks after transplant byin vivo bioluminescent imaging and human CD34+/CD45+, CD2+/CD7+/CD45+ cells were found in hematopoietic organs of engrafted mice at 8 weeks post transplant. Importantly, the engraftment of CD34+/CD45+, CD2+/CD7+/CD45+ cell populations in hematopoietic organs derived from T-ALL patient samples correlates with the status of mutations. Finally, isolated human CD34 progenitor cells could engraft 2nd and 3rd recipients demonstrating their propensity for self-renewal and differentiation. 1. Enhanced Notch1 expression was found in T-ALL CD34+ cells. 2. Candidate T-ALL LSC (CD34+/CD4−/CD7−) give rise to CD34+/CD45+ and CD2+/CD7+/CD45+ cells in the hematopoietic organs of transplanted mice. 3. T-ALL LSC can be tracked in 1st and 2nd transplanted mice providing a robust in vivo model for testing novel self-renew pathway inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4313.

Abstract LB-258: Smoothening the way for human leukemia stem cell inhibition in chronic myeloid leukemia

Chronic myeloid leukemia (CML) progresses from a chronic phase (CP) state that harbors the BCR-ABL fusion gene, to CML blast crisis (BC), characterized by activation of -catenin within granulocyte-macrophage progenitors (GMP) (Jamieson et al, NEJM 2004). BC CML GMP serially transplant leukemia in vivo and form myeloid sarcomas (Abrahamsson et al, PNAS 2009). Wnt, GSK3 and Shh family molecules are implicated in driving leukemia progression and because GSK3 is a negative regulator of both Wnt and Shh signaling pathways, we evaluated whether GSK3 deregulation activates -catenin and Gli simultaneously. We investigated whether treatment with a SMO antagonist in combination with dasatinib decreases human BC CML engraftment in vivo. BC CML tumor cells were treated for 0 and 7 days in vitro with vehicle (DMSO) or SMO antagonist (Pfizer; PF-04449913) and harvested for Q-PCR analysis. FACS-purified BC CML progenitors (CD34+CD38+Lin-PI−) were transplanted intrahepatically into newborn RAG2−/− c−/− mice and serially transplanted for propagation of tumors. Harvested tumors were then CD34-selected and transplanted to tertiary recipients (1×105 cells per mouse). Mice were treated for 14 days starting at 8 weeks of age. Mice were dosed once daily by oral gavage with 200µl of drug or vehicle (50% PEG in HBSS). The treatment included PF-04449913 (100mg/kg) with or without dasatinib (50mg/kg). After treatment the percentage of LSC (CD34+CD38+CD45RA+CD123+Lin-PI−) was determined by FACS analysis of the bone marrow and liver and number of tumors evaluated. BC CML progenitors harbors an up regulation of Gli mRNA and a downregulation of Ptch1 mRNA (n=3) compared to normal cord blood. Gli mRNA expression decreases following in vitro treatment with PF-04449913. Treatment with PF-04449913 alone in human BC CML LSC transplanted mice (n=3 exp., n=15 mice) does not significantly inhibit engraftment compared with vehicle (n=3 exp., n= 27 mice). Although dasatinib alone decreases tumor formation (P≤0.05), it does not eradicate the LSC in the liver or bone marrow (n=3 exp., n=20 mice). Combination treatment with PF-04449913 and dasatinib revealed a significant decrease in LSC engraftment in livers (P≤0.05) compared with the PF-04449913 or dasatinib alone. The combination treatment completely eradicated the LSC capability of tumor formation (0.0) in secondary recipients (n=5) compared with dasatinib that had an average of 1.8 tumors (n=6), PF-04449913 that had an average of 4.9 (n=7) and vehicle group that had 3.8 tumors (n=13). Colony formation assays were performed with cord blood CD34+ progenitors (n=3 samples) and no toxicity could be seen with PF-04449913 (1µM) compared with vehicle. There was no significant inhibition of normal CD34+ colony replating following PF-04449913 compared with vehicle treatment. A phase I clinical trial with PF-04449913 in combination with dasatinib is currently being executed for advanced phase CML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-258.