ALBERT

Description: The myeloproliferative neoplasms (MPNs) are hematologic malignancies with a chronic clinical course and a risk of developing thrombosis and acute leukemia. JAK2 is a member of the Janus family of non-receptor tyrosine kinases, and the somatic activating point mutation of JAK2 (JAK2V617F) has been found in Philadelphia chromosome-negative MPNs in approximately 95% of polycythemia (PV), 60% of essential thrombocytosis (ET), and 50% of JAK2 V617F-positive MPD (PMF) patients. Although several JAK2 inhibitors are in the early stage of clinical trials and have been shown to help to improve symptoms and quality of life in patients, their long-term effectiveness in patients remains to be determined. New therapeutic strategies need to be developed for treating PV more effectively. Because hematopoietic stem cells (HSCs) harbor JAK2V617F in PV patients, PV should be compared to Philadelphia chromosome-positive chronic myeloid leukemia (CML) that is also derived from HSCs and has a myeloproliferative phenotype similar to PV in mice. Therefore, we reasoned that a gene essential for CML development might also play a critical role in PV development. We have shown that the survival and self-renewal of CML-initiating cells require the arachidonate 5-lipoxygenase gene (Alox5) and that Alox5 is essential for CML development. Therefore, in this study we investigated the role of Alox5 in pathogenesis of PV in mice. We showed that JAK2V617F upregulates Alox5 expression both in vitro (Ba/F3 cells) and in vivo (the spleen cells in PV mice). To examine whether Alox5 is required for induction of PV by JAK2V617F, we transduced bone marrow (BM) cells from wild type (WT) or Alox5 homozygous knockout (Alox5-/-) mice with JAK2V617F-GFP, followed by transplantation into lethally-irradiated recipient mice. We found that the average percentage of JAK2V617F-expressing white blood cell counts (GFP+ WBCs) in recipients of JAK2V617F-transduced Alox5-/- donor BM cells was significantly lower than that in recipients of JAK2V617F-transduced WT donor BM cells (P〈 0.001). Total numbers of WBCs, red blood cells (RBCs), hemoglobin (HGB) and hematocrit (HCT) were also significantly lower in the absence of Alox5. With time (at 9 months after induction of PV), the effect of Alox5 deficiency on PV development became more significant. Because JAK2V617F-transformed HSCs is shown to be a PV-initiating cell population, we further tested whether loss of Alox5 affects this cell population in PV mice. We found that the percentage and total number of HSCs (Lin-Sca-1+c-Kit+) in mice receiving JAK2V617F-transduced Alox5-/- BM cells was significantly lower than that in mice receiving JAK2V617F-transduced WT BM cells. These findings suggest that Alox5 could be an effective target gene for PV. We tested this idea by treating PV mice with an Alox5 inhibitor zileuton (300 mg/kg, once a day) or a placebo. Western blot analysis of protein lysates from the spleens of PV mice showed that 5-lipoxygines (5-LO, a protein product of the Alox5 gene) is upregulated by JAK2V617F, and inhibition of Alox5 function by zileuton restored the 5-LO level back to the control level. In addition, the number of WBCs in placebo-treated mice rose significantly with time, whereas the number of WBCs in zileuton-treated mice did not. The inhibition of PV development by zileuton correlated with much smaller infiltrated spleen in zileuton-treated PV mice than in placebo-treated PV mice. Furthermore, we compared the levels of JAK2V617F-expressing cells (GFP+) in PV mice. FACS analysis showed that the percentage of Gr-1+ or Mac+ PV cells in peripheral blood was significantly lower in zileuton-treated PV mice than in placebo-treated PV mice. Consistent with the inhibition of PV cells by zileuton in mice, survival of zileuton-treated PV mice was significantly improved compared to placebo-treated PV mice (Fig. 3I). At 5 months after induction of PV, about 40% placebo-treated PV mice died, but all zileuton-treated mice survived. To understand the underlying mechanisms, we treated JAK2V617F-expressing BaF/3 cells with zileuton, and found that zileuton inhibited activation of AKT and β-catenin by JAK2V617F, and did not affect activation of PI3K, MAPK and JAK2. Together, these results demonstrate that Alox5 represents a critical pathway in JAK2V617F-induced PV and that targeting of Alox5 provides a new strategy for treating PV. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 188 We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice (Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia. Nature Genetics 41:783-792, 2009). We believe that the Alox5 pathway represents a major molecular network in LSCs. Therefore, we decided to further dissect this pathway by comparing gene expression profiles between wild type and Alox5−/− LSCs from CML mice using the DNA microarray analysis. We identified a small group of candidate genes that were changed in expression in the absence of Alox5. Among these genes, we have identified the Msr1 gene and chosen to test the function of this gene in regulating LSC function, because this gene was up-regulated, indicating that it might play a tumor suppressor role in LSCs. In our CML mouse model, we observed that recipients of BCR-ABL transduced Msr1−/− bone marrow cells developed CML much rapidly than recipients of BCR-ABL transduced wide type bone marrow cells. To test whether this accelerated CML is related to abnormal function of LSCs, we carried out a serial transplantation assay by transferring bone marrow cells from primary recipients of BCR-ABL-transduced wild type or Msr1−/− donor bone marrow cells into secondary and next-generation of recipient mice to biologically assess the effect of Msr1 on LSCs. BCR-ABL-expressing wild type leukemia cells from bone marrow of CML mice were only able to transfer CML once, whereas BCR-ABL-expressing Msr1−/− leukemia cells were able to transfer lethal CML for five genrations. This observation indicates that BCR-ABL-expressing Msr1−/− LSCs have markedly increased stem cell function. To further compare the stem cell function, we performed the leukemia stem cell competition assay by 1:1 mixing wild type (CD45.1) and Msr1−/− (CD45.2) bone marrow cells from CML mice. At day 25 or 30 after transplantation, more than 60% and 95% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2+Msr1−/− myeloid leukemia cells, and all these mice developed CML and died of CML derived from Msr1−/− LSCs. To confirm the tumor suppressor role of Msr1 in CML development, we co-expressed BCR-ABL and Msr1 in MSR1−/− bone marrow cells by retroviral transduction, followed by transplantation of these cells into recipient mice. The ectopically-expressed Msr1 in MSR1−/− bone marrow cells rescued the accelerated CML phenotype, and some recipient mice did not even develop the CML. Together, these results demonstrate that Msr1 plays a tumor suppressor role in LSCs. The Msr1 pathway is a novel molecular network in LSCs, and it will be important to fully study this pathway for developing curative therapeutic strategies for CML. Disclosures: No relevant conflicts of interest to declare.

Description: The Abl tyrosine kinase inhibitors (TKIs) imatinib mesylate (IM) and dasatinib, targeting BCR-ABL for the treatment of Philadelphia-positive (Ph+) leukemia including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL), have produced impressive results in terms of therapeutic outcome and safety for patients. However, clinical resistance to these TKIs likely at the level of leukemic stem cell negates curative results in Ph+ leukemia. At present, an anti-stem cell strategy has not been developed for treating these leukemia patients. Homoharringtonine (HHT) (omacetaxine mepesuccinate - USAN/INN designation) has shown significant clinical activity in CML in combination with IM or alone for patients failing IM. However, little is known about whether HHT has an inhibitory effect on leukemic stem cells. The purpose of this study is to determine whether HHT inhibits BCR-ABL-expressing leukemic stem cells (Lin-c-Kit+Sca-1+) that we identified previously (Hu et al. Proc Natl Acad Sci USA 103(45):16870–16875, 2007) and to evaluate therapeutic effects of HHT on CML and B-ALL in mice. We find that in our in vitro stem cell assay, greater than 90% of leukemic stem cells were killed after being treating with HHT (12.5, 25, and 50 nM) for 6 days, and in contrast, greater than 75% or 92% of leukemic stem cells survived the treatment with dasatinib (100 nM) or imatinib (2 mM). We next treated CML mice with HHT (0.5 mg/kg, i.p., once a day). 4 days after the treatment, FACS analysis detected only 2% GFP+Gr–1+ myeloid leukemia cells in peripheral blood of HHT -treated CML mice and in contrast, 41% GFP+Gr–1+ myeloid leukemia cells in placebo-treated mice. We also treated mice with BCR-ABL induced B-ALL with HHT, and found that only 0.78% GFP+B220+ lymphoid leukemia cells were detected in peripheral blood compared to 34% GFP+B220+ lymphoid leukemia cells in placebo-treated mice. Furthermore, HHT significantly inhibited in vitro proliferation of K562 and B-lymphoid leukemic cells isolated from mice with B-ALL induced by BCR-ABL wild type and BCR-ABL-T315I resistant to both imatinib and dasatinib. In sum, HHT has an inhibitory activity against CML stem cells, and is highly effective in treating CML and B-ALL induced by BCR-ABL in mice.

Description: Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) remains challenge for the treatment of chronic myeloid leukemia (CML). IM resistance often results from unknown mechanisms with wild type BCR-ABL that have no effects on TKIs binding to ABL kinase domain. The basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed an initial bioinformatics screen on over represented CML genes, followed by a quantitative PCR screen of genes that were elevated in TKIs resistant CML samples. We identified a total of 33 candidate genes that were highly expressed in TKIs resistant patients. Among these genes, 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3) controlling the limiting step of glycolysis, was found to strongly associated with TKIs resistance. RNA interference of the expression of PFKFB3 and pharmacological inhibition of its kinase activity markedly increased the sensitivity of TKIs resistant CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 prevented CML cells growth and significantly improved the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment demonstrated that the Ets transcription factor PU.1 regulates the elevated expression of PFKFB3 in TKIs resistant CML cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent TKIs resistant CML. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1672 Chronic Myelogenous Leukemia (CML) is a myeloproliferative neoplasm (MPN) caused by transformation of hematopoietic stem cells (HSCs) by the BCR-ABL oncogene. CML is associated with excessive proliferation of HSCs and massive expansion of the myeloid cell pool. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPα, along with various GABPβ partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. In a mouse model of CML, animals transplanted with BCR-ABL-infected bone marrow developed massive myeloid cell expansion and died with a MPN. Induced deletion of Gabpa prevented development of CML, yet mice continued to produce mature BCR-ABL-expressing granulocytes for months without apparent illness. BCR-ABL+ cells were transplantable into secondary recipients without development of CML, and contributed to all hematopoietic lineages, thereby confirming expression of BCR-ABL by long-term HSCs. We used a bioinformatic approach to analyze GABP-bound genes that are upregulated in both human and mouse LSCs compared to normal HSCs. Among 115 GABP-bound, CML-associated genes, we identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase that has been implicated in cancer, but has not previously been associated with HSC functions or CML. Deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In vitro growth of BCR-ABL+ bone marrow cells was prevented by Gabpa deletion, but growth was partially rescued by forced expression of PRKD2. Knockdown and pharmacologic inhibition of PRKD2 blocked cell cycle entry of BCR-ABL+ cells. We conclude that Gabp is required for HSC cell cycle entry and for development of CML, and that these effects of GABP are mediated, in part, by PRKD2. These findings suggest that PRKD2 kinase may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1666 NF-κB activation has been linked to the promotion of an assortment of malignancies. While in vitro studies have supported a tumor-promoting role for NF-κB in leukemias, in vivo evidence has not been collected. NF-κB has also been proposed as a therapeutic target for Philadelphia chromosome-positive leukemias including chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). In this study, we investigate the role the Nfkb1 gene plays in CML and B-ALL using mouse models for these leukemias induced by the BCR-ABL oncogene. Contrary to conventional thinking, we find a tumor-suppressing role for Nfkb1 in BCR-ABL leukemias. To induced CML, we transduced bone marrow cells from 5-FU-primed wild type or Nfkb1−/− mice with BCR-ABL-GFP retrovirus, followed by transplantation of the transduced cells into lethally irradiated recipient mice. We show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells developed CML more rapidly than the wild type recipients, correlating to elevated percentages (82.3% vs 38.5%) and total numbers of Gr-1+GFP+ cells in peripheral blood of the mice. We next tested whether the loss of Nfkb1 also accelerated B-ALL development under the B-ALL-inducing conditions, and show that recipients of BCR-ABL-transduced Nfkb1−/− donor cells succumbed to B-ALL more quickly than the wild type recipients. These results suggest that Nfkb1 does not stimulate leukemia growth, and instead, it plays a tumor-suppressing role in CML and B-ALL. Because these findings are opposite from what people believe in the field, we confirmed our findings by conducting a rescue experiment. The Nfkb1 gene encodes the p105 protein; roughly half of p105 product is selectively proteolyzed to yield the p50 transcription factor. In this rescue experiment, we transduced Nfkb1−/− donor cells with BCR-ABL alone or BCR-ABL and p50 of Nfkb1 to induce leukemia, and we show that acceleration of leukemia development caused by the Nfkb1 loss is partially reversed by p50. To study the underlying mechanism, we focused on CARD11, because it plays a critical role in mediating NF-κB signaling in lymphocytes. We find that CARD11 expression is induced in myeloid and increased in lymphoid cell lines expressing BCR-ABL. Furthermore, we induced B-ALL using Card1−/− mice. We find that similar to the loss of Nfkb1, the loss of Card11 expression also accelerates the development of B-ALL. To increase human relevance of our findings, we overexpressed p50 in human K562 cells by retroviral transduction with p50-IRES-GFP or empty vector, and find that FACS-sorted p50/GFP K562 cells grow significantly slower than empty vector-transduced cells. These findings support an idea that Nfkb1 signaling suppresses BCR-ABL-induce leukemia at least partially through a CARD11-dependent mechanism. In summary, our results demonstrate that Nfkb1 plays a tumor-suppressing role in BCR-ABL-induced leukemias, arguing that we should be careful when targeting of Nfkb1 in leukemia treatment. Disclosures: No relevant conflicts of interest to declare.

Description: The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However, it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome–positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL–induced leukemias, we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition, overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover, PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL–induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome–positive leukemia.