Blast Crisis Leukemia Stem Cells Research Articles

Abstract 2134: Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients

Abstract The marketed BCR-ABL tyrosine kinase inhibitor (TKI), imatinib (Gleevec™) is a very successful targeted anti-cancer therapy. It has revolutionized the treatment of early stage or chronic phase (CP) chronic myeloid leukemia (CML). Unfortunately, a proportion of CP patients experience suboptimal responses to BCR-ABL TKIs, and progress to blast crisis (BC) stage of CML with poor survival rate. A potential cause of the resistance to TKI is the elevated level of phosphorylated eukaryotic initiation factor 4E (eIF4E), which has been found to be a consistent feature in patient-derived BC-CML samples. Importantly, both in vivo and in vitro studies have demonstrated that the MAP kinase-interacting serine/threonine-protein kinases 1 and 2 (MNK1/2) phosphorylate eIF4E on Ser209, and that the overexpression of eIF4E drives oncogenesis in a variety of cancers including BC-CML. Furthermore, several reports have indicated that eIF4E phosphorylation at Ser209, as well as eIF4E overexpression, is critical to tumor progression. We found that a BC-CML cell line, K562, that expresses a serine to alanine phospho-mutant at position 209 of eIF4E, shows reduced ability to form tumors in mice compared to wildtype eIF4E. In addition, our recent work has demonstrated the importance of the MNK-eIF4E axis in activating BC leukemia stem cell (LSC) function (Lim et al., PNAS18; 110(25):E2298-307, 2013). These data highlight the critical importance of MNK1/2-dependent eIF4E phosphorylation in cancer progression and maintenance, and suggests that inhibition of MNK1/2 is an attractive therapeutic approach to treat BC-CML. Consequently, we set out to identify selective inhibitors of the MNK1/2 kinases to treat BC-CML patients. Here, we report our hit finding strategy, as well as our hit to lead optimization process. Results describing structure activity relationships, pharmacokinetics properties, and biochemical characteristics of a highly specific MNK1/2 inhibitor, are presented. Our data demonstrate that drug-like molecules can be developed to potently and specifically inhibit the MNK kinases. We also show that simultaneous inhibition of MNK and BCR-ABL is effective at inhibiting BCR-ABL-driven growth and proliferation, as well as inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs. A combination of selective MNK and BCR-ABL inhibitors may provide clinical benefit to BC-CML patients. Citation Format: Kassoum Nacro, Haiyan Yang, Melvyn Wai Tuck Ho, Yoon Sheng Yeap, Lohitha Rao Chennamaneni, Shi Hua Ang, Eldwin Sum Wai Tan, Athisayamani Jeyaraj Duraiswamy, Sharon Lim, Boping Liu, Esther Hongqian Ong, Meng Ling Choong, Shi Jing Tai, Vithya Manoharan, Vishal Pendharkar, Lijun Ding, Yun Shan Chew, Joma Kanikadu Joy, John LW Kuan, Perlyn Z. Kwek, Anders Poulsen, May Ann Lee, Kanda Sangthongpitag, Charles Chuah, Tiong S. Ong, Jeffrey Hill, Thomas H. Keller, Alex Matter. Rational design of selective MNK 1 and 2 kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2134.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0453-9) contains supplementary material, which is available to authorized users.

Malignant Reprogramming of Progenitors into Leukemia Stem Cells Is Enhanced By Upregulation of CD44 Transcript Variant 3 in Malignant Microenvironments

IntroductionMalignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche.Methods and ResultsCD44 Isoform Expression AnalysisTo investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34+CD38+Lin-) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency.Lentiviral CD44 Variant 3 OverexpressionTo directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency.Targeted Inhibition of CD44 variant 3 Expressing LSCHumanized RAG2-/-gc-/-mice engrafted with CD34+ BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression.ConclusionsUpregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. DisclosuresNo relevant conflicts of interest to declare.

Effective and Selective Elimination of CML Stem Cells Using Novel Ethacrynic Acid Derivatives

Tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML but do not eliminate primitive, quiescent leukemia stem cells (LSC), which persist as a potential source of leukemia relapse. Additional strategies to enhance eradication of LSC are required to increase the possibility of treatment free remissions for CML patients. Glutathione S-transferase P1-1 (GSTP1-1) is a Phase II detoxifying enzyme which is overexpressed in several cancers and causes drug resistance. The diuretic Ethacrynic acid (EA) is a GSTP1-1 activity inhibitor and also induces celldeath in malignant cells at high concentrations. We have synthesized two EAoxadiazole analogs, 6U and 6S, which demonstrate enhancedpro-apoptotic effects in CML K562 cells expressing high levels of GSTP1-1. Previously we found that 6U and 6S induced apoptosis through downregulation of anti-apoptotic protein MCL-1 in addition to their GSTP1-1 activity inhibition. We extended these observations to primary CML stem/progenitor cells. We observed increased expression of GSTP1-1 mRNA and protein, associated with increased expression of MCL-1, BCL2 and BCL-xL, in chronic phase (CP) and blast crisis (BC) CML compared to normal CD34+ cells. Treatment of CP CML CD34+ cells with 6U or 6S (1 to 6µM) for 24 to 48 hours resulted in a significant dose-dependent induction of apoptosis, inhibition of cell growth, and reduction in colony forming cell (CFC) numbers, with 6U demonstrating greater efficacy than 6S. Treatment with 6U did not induce significant apoptosis of normal (NL) CD34+ cells at doses below 4µM. 6U induced significantly less apoptosis in NL compared with CML CD34+ cells (2µM, p<0.05). We further tested the activity of 6U against purified CML and normal CD34+CD38- stem/primitive progenitors and CD34+CD38+ committed progenitors with or without the BCR-ABL TKI Dasatinib (DAS). 6U treatment induced apoptosis of CML, but not normal, CD34+CD38- and CD34+CD38+ cells (Table). Combination of 6U with DAS (50nM) selectively enhanced apoptosis of CML compared to normal cells, including quiescent, slowly dividing CML LSC that are resistant to TKI-induced apoptosis (p≤0.01). Treatment with 6U alone or with DAS, significantly increased G1, and decreased S/G2/M phase of CML, but not in normal CD34+ cells, and reduced CFC growth from CML CD34+CD38+ cells (Table). CML, but not normal CD34+ cells, treated with 6U, with or without DAS, prior to transplant, showed significantly reduced engraftment in NSG mice, indicating selective inhibition of in vivo repopulating CML LSC (Table). Treatment with 6U was also effective in inducing apoptosis and inhibiting CFC growth in BC CML progenitor cells (Table). 6U treatment resulted in down-regulation of GSTPI1-1 and MCL-1 protein expression in CP and BC CML, but not in normal CD34+ cells. Interestingly 6U treatment also reduced BCR-ABL protein expression in CP and BC CML CD34+ cells. We conclude that CML CP and BC LSC express high levels of GSTP1-1 and anti-apoptotic proteins, which can be targeted by the novel EA derivative 6U through a new mechanism. Since 6U has significantly lesser effects on normal stem cells, it may offer a promising and innovative approach to selectively target CP and BC CML LSC in combination with TKI inhibitors.Abstract 4508. TableCML CPNormalCML BCCtrl6UDASDAS+ 6UCtrl6UDASDAS+ 6UCtrl6UDASDAS+ 6UApoptosis (normal, CP CML: CD34+CD38-; CML BC CD34+)3.4± 0.9 15.9±6.7 9.4± 2.6 47.4±13.6 **3.3± 0.9 5.1± 1.0 1.6± 0.2 7.0± 1.2 *3.4± 0.7 30±12.7 10.6±1.8 43.3±14.1 **CFU-GM (normal, CP CML: CD34+CD38+; CML BC CD34+)71.3± 7.8 7± 3.2 **21± 7.3 **5 ± 2.3 **121±19.3 102.7±6.2 134.3±15.9 103±5.1 288.5±89.4 26.5±11.3 ***82.7±33.1 **8 ± 3.6 ***NSG engraftment (CD34+)1.8± 0.30.4± 0.1 ***0.8± 0.3 **0.4± 0.04 ***68.2± 4.961± 2.268.1± 2.964.2± 3.9Data shown are mean ± SEM of 3-6 samples. Significance, compared to controls. *p≤0.05,**p≤0.01, ***p≤0.001 DisclosuresNo relevant conflicts of interest to declare.

Abstract 1912: ADAR1-mediated microRNA regulation and blast crisis leukemia stem cell generation in chronic myeloid leukemia

Abstract Introduction Chronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSC). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis (BC) transformation, BC LSC generation and TKI resistance. We have recently shown that adenosine to insoine (A-to-I) RNA editing activity, mediated by the adenosine deaminase acting on dsRNAs ADAR1, is enhanced in BC CML and drives leukemic progression by inducing myeloid progenitor reprogramming and enhancing self-renewal. ADAR1 targets double-stranded RNA hairpin-containing loop structures, such as microRNAs (miRNAs). MiRNAs, by operating through base-pairing with complementary sequences within a mRNA molecule lead to mRNA degradation and gene silencing. Emerging evidence suggests miRNAs alterations introduced at the pri-miRNA or pre-miRNA level by A-to-I editing by ADAR1 may be responsible for broad changes in the transcriptome that drives LSC maintenance. However, the overall change in edited miRNA profiles in CP CML compared with BC progenitors is unclear. In this study we aimed to investigate the role of ADAR1 in driving malignant progression by focusing on its regulation of miRNA expression in CML. Methods and Results MicroRNA expression profiles were evaluated in CD34+ cells derived from CP and BC CML patient samples as well as cord blood. MiRNA PCR array (Qiagen) allowed us to identify differentially expressed miRNAs that were subsequently validated by q-PCR with specific primers targeting individual miRNA. We observed the downregulation of 13 miRNA in CML BC compared to CML CP progenitors. Lentiviral overexpression of ADAR1 in CD34+ cord blood cells led to significant downregulation of 17 miRNAs, including members of the let-7 family, and other miRNAs involved in the regulation of self-renewal. Interestingly, BC CD34+ cells showed a miRNA profile similar to that of lentiviral ADAR1 transduced cord blood progenitors. Lentiviral overexpression of ADAR1 in CD34+ chronic phase CML cells, led to a significant downregulation of 14 miRNAs compared with their lentiviral backbone transduced counterparts. Conclusion Our data suggest that ADAR1 contributes to BC LSC generation by downregulating miRNAs that target stem cell regulatory gene products. A further characterization of ADAR-editing related miRNA signatures may enable BC LSC detection and early intervention with combined BCR-ABL1 and ADAR1-targeting strategies that may obviate CML relapse and progression. Citation Format: Maria Zipeto, Qingfei Jiang, Leslie Crews Robertson, Catriona HM Jamieson. ADAR1-mediated microRNA regulation and blast crisis leukemia stem cell generation in chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1912. doi:10.1158/1538-7445.AM2014-1912

Abstract 4798: The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation

Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm initiated in hematopoietic stem cells by expression of the BCR-ABL1 fusion oncogene and its protein product, which enhances ABL1 kinase activity. Targeted BCR-ABL tyrosine kinase inhibitors (TKIs), such as Imatinib, and the second generation TKIs like Nilotinib as well as the dual specific SCR and ABL inhibitor Dasatinib have significantly slowed disease progression by eradicating the bulk of BCR-ABL1 expressing cells in the circulation. However, progression to a therapeutically resistant blast crisis (BC) phase is driven by CD34+CD38+Lin- progenitors that co-opt stem cell properties, such as enhanced self-renewal and survival, albeit in a deregulated manner. These BC leukemia stem cells (BC LSC) harbor enhanced BCL2 and beta-catenin expression. Seminal research shows that a key Wnt/beta-catenin target gene, CD44, plays a vital role in cancer stem cell survival and retention in the malignant microenvironment. Because alternative mRNA splicing in humans generates many CD44 isoforms, we investigated CD44 variant expression and retention of human BC LSC in specific hematopoietic niches. Methods and Results We performed whole transcriptome RNA sequencing (RNA Seq) of FACS sorted progenitors (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8) CML patients as well as from normal cord blood (CB) (n=7) and adult peripheral blood (NPB)(n=4). A number of CD44 transcript variants were detected: 3, 4 (CD44s), 5, 6, 7, 8 plus additional variants. RNA seq analysis uncovered a higher overall CD44 expression in BC compared to CP progenitors. Notably, one isoform of CD44, variant 3, was highly upregulated in BC compared to CP progenitors and minimally expressed by their normal counterparts. Moreover, BC progenitors that engrafted in RAG2-/-γc-/- harbored CD44v3 expression in the bone marrow and the splenic niche and this differential BC LSC isoform expression was reduced after dasatinib treatment. In addition, BC LSC in the spleen showed a reduction of migration specific markers, such as RHAMM, ICAM-1 and Osteopontin, compared with the bone marrow resident BC LSC. Furthermore, a comparative splice isoform specific qRT-PCR analysis of CD44 variants expression in young versus old bone marrow showed no correlation with aging. In fact, human embryonic stem cells harbored variant 3 expression. Conclusions These data suggest that CD44 transcript variant 3 upregulation occurs following malignant reprogramming of hematopoietic progenitors that enables them to adopt features of an embryonic transcriptional program in select microenvironments. Detection of CD44 variant 3 has the potential to more precisely identify patients at risk for relapse and progression, suggest that combined therapeutic strategies involving a BCR-ABL specific TKI and a CD44 monoclonal antibody may decrease relapse and BC transformation rates. Citation Format: Frida L. Holm, Eva Hellqvist, Cayla Mason, Christian Barrett, Kelly A. Frazer, Anil Sadarangani, Catriona HM Jamieson. The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4798. doi:10.1158/1538-7445.AM2014-4798

Inhibition Of Inflammation Driven Leukemia Stem Cell Self-Renewal With a Selective JAK2 Antagonist

Chronic myeloid leukemia (CML) represents an important paradigm for identifying the molecular events that promote malignant reprogramming of progenitors into therapeutically recalcitrant leukemia stem cells (LSC) during blast crisis (BC) transformation. To elucidate mechanisms of human BC LSC generation, whole transcriptome RNA sequencing (RNA Seq), lentiviral BCR-ABL and JAK2 transduction, quantitative RT-PCR (qRT-PCR) and serial xenotransplantation studies were performed. In human BC LSC, RNA seq revealed extensive upregulation of inflammation-responsive genes in conjunction with JAK/STAT signaling pathway activation and splice isoform specific qRT-PCR uncovered a predilection for selective STAT5a isoform expression. While lentiviral BCR-ABL1 expression in cord blood progenitors enhanced JAK2 activation and expression of specific STAT5a splice isoforms, lentiviral human JAK2 overexpression globally activated inflammation-response genes and expression of adenosine deaminase RNA associated (ADAR1), a primate specific RNA editase previously shown to activate self-renewal in response to inflammation. Notably, inhibition of BC LSC self-renewal with dasatinib, a BCR-ABL inhibitor, combined with a potent JAK2 inhibitor, SAR302503, was associated with reduced STAT5a isoform expression and phospho-STAT5 activation as well as ADAR1 expression and activity. These results highlight a novel JAK/STAT pathway driven niche-responsive mechanism of human BC LSC generation that can be targeted, at least in part, with a selective JAK2 inhibitor and may be utilized as an RNA editing-based biomarker of cancer stem cell generation and therapeutic resistance. Disclosures:Jamieson:Sanofi: Consultancy.

Dual Specific Inhibitors Of The BCR-ABL and MNK Kinases As Potential Therapeutics For Blast Crisis Chronic Myeloid Leukemia

Pharmacologic inhibition of BCR-ABL by tyrosine kinase inhibitors (TKI) provides effective therapy for chronic phase but not blast crisis (BC) CML. The inability of TKIs to control BC CML is due to the reactivation of BCR-ABL through TKI resistance-conferring mutations, as well as the activation of alternative oncogenic pathways that mediate acquired leukemia stem cell (LSC) function and differentiation block in leukemic progenitors. Accordingly, the development of effective BC therapeutics will require reversing multiple contributors to the BC phenotype, and indicates that targeting BCR-ABL alone will not be sufficient to control BC CML. Indeed, despite the ability of third-generation TKIs to effectively inhibit the majority of clinically-relevant BCR-ABL kinase domain mutations, most patients with BC treated with these agents continue to relapse despite initial responses. Because our recent work has highlighted the importance of the MNK kinase-eIF4E axis in b-catenin-associated BC self-renewal (Lim et al., PNAS18;110(25):E2298-307, 2013), we set out to develop compounds capable of targeting BCR-ABL, TKI-resistant BCR-ABL mutants, as well as the MNK kinases simultaneously. Here, we report the discovery and characterization of two lead compounds ETC-219 and ETC-027 that inhibit clinically relevant BCR-ABL isoforms (E255K, T315I) as well as the MNK1/2 kinases at nanomolar potency (Table 1).Table 1In vitro kinase assays for ETC-219 and ETC-027.Compound IDIC50 MNK1 (µM)IC50 MNK2 (µM)IC50 ABL (µM)IC50 ABL (T315I) (µM)IC50 ABL (E255K) (µM)ETC-2190.2540.0150.0140.0130.148ETC-0275.0150.0240.2140.0913.19Using a panel of 104 kinases, we showed that ETC-219 and ETC-027 inhibited 90% of the activity of 15 and 12 kinases respectively when tested at 1uM. ETC-219 and ETC-027 interact with only 27 and 20 of off-target kinases respectively, and offer significant selectivity over kinases outside of the MNK and BCR-ABL families.ETC-219 and ETC-027 have favorable pharmacokinetic properties, and include the following ADME characteristics (Table 2).Table 2ADME characteristics of ETC-219 and ETC-027.ETC-219ETC-027ParametersUnitIVPOIVPODosemg/kg1050550C0ng/ml21241714Cmaxng/ml12122490Tmaxh12T1/2h22.21.82.1CLL/h/kg39.72.83.5VzL/kg8.831.37.410.8VssL/kg7.25.8AUC0-t(last)ng.h/ml32905128179514337AUC0-infng.h/ml33025133179814349Extrapolated area for AUC0-inf%0.37000.11000.16000.0800F%3180Regarding targeting of BCR-ABL-dependent cellular proliferation and survival, we found that ETC-219 and ETC-027 exhibited potent anti-proliferative activity against a panel of BC CML cell lines (K562, BV-173, EM-2, KCL-22, JURL-MK1), as well as Ba/F3-BCR-ABL cells containing the T315I mutation. In an in vivo xenograft model, employing K562-eIF4E cell lines in NOD-SCID mice, we found that both compounds inhibited tumor growth in a dose-dependent manner, and a dose-dependent decrease in eIF4E phosphorylation in tumor explants were also observed. Tumor regression was observed at 100 mg/kg for ETC-219. Importantly, mice given ETC-219 and ETC-027 at 100 mg/kg and 200 mg/kg doses did not exhibit significant toxicity.With respect to MNK-eIF4E-b-catenin axis inhibition, ETC-219 and ETC-027 inhibited eIF4E phosphorylation at IC50s of 0.186 nM and 0.28 nM respectively in HeLa cells. In parallel, both compounds inhibited Wnt/b-catenin reporter activity in BC CML cell lines, as well as the expression of a panel of Wnt-regulated genes. Using the serial replating assay as a readout for LSC function and self-renewal capacity, we found that ETC-219 and ETC-027 were effective at 1nM at preventing serial replating using primary CD34+ cells from patients in BC. Furthermore, ETC-219 and ETC-027 inhibited the serial replating efficacy of BC cells from patients with clinical resistance to the third generation TKI, ponatinib. ConclusionOur results demonstrate that: i) small molecule compounds with favorable ADME properties can be developed to potently and specifically inhibit the BCR-ABL and MNK kinases simultaneously; ii) Dual specific MNK and BCR-ABL inhibitors are effective at abrogating BCR-ABL-driven growth and proliferation, as well as the MNK-eIF4E-dependent self-renewal function of BC LSCs. We conclude that dual specific BCR-ABL and MNK inhibitors may warrant testing in patients with BC CML. Disclosures:Chuah:Novartis: Honoraria; BMS: Honoraria.

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

The RNA Editase ADAR1 Promotes Malignant Progenitor Reprogramming in Chronic Myeloid Leukemia.

AbstractAbstract 2772While advanced malignancies in Chronic Myeoloid Leukemia (CML) are diverse in phenotype, they often exhibit stem cell properties including enhanced survival, quiescence and self-renewal potential. The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. While DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity may also be generated by aberrant RNA editing mediated by adenosine deaminase acting on dsRNA (ADAR) family, which have been shown to promote an embryonic transcriptional program and regulate fetal and adult hematopoietic stem cell (HSC) self-renewal as well as stem cell responses to inflammation. In this study, whole transcriptome sequencing of normal, chronic phase (CP) and functionally validated blast crisis (BC) chronic myeloid leukemia (CML) progenitors revealed increased inflammatory pathway gene expression in concert with BCR-ABL amplification, enhanced expression of interferon-responsive ADAR1 and a propensity for increased A-to-I RNA editing during CML progression. Mechanistic studies demonstrated that lentivirally enforced ADAR1 p150 expression induced expression of the myeloid-skewing transcription factor PU.1 and skewed cell fate towards granulocyte-macrophage progenitors - the initiating LSC population in BC CML. Moreover, lentiviral ADAR1 knockdown reduced BC LSC self-renewal capacity in RAG2−/−gc−/− mice. These data shed new light on the role of ADAR1-directed RNA editing in myeloid progenitor reprogramming and self-renewal potential of malignant progenitors that drive disease progression and therapeutic resistance in CML, and provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies. Disclosures:No relevant conflicts of interest to declare.

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

Abstract 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

Cycling Toward Leukemia Stem Cell Elimination Wtih a Selective Sonic Hedgehog Antagonist,

Abstract 3776Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse. Disclosures:Levin:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.

Combination Targeted Therapy to Impair Self-Renewal Capacity of Human Blast Crisis Leukemia Stem Cells

Abstract 1693The aim of this study is to develop clinical strategies that will HALT progression of CML by reducing leukemia stem cell (LSC) burden using a clinical grade JAK2 inhibitor, SAR302503 (SAR503, Sanofi, Cambridge, MA), alone or in combination with a potent BCR-ABL inhibitor, dasatinib.For this, CML patient samples in blast crisis phase (BC CML) were subjected to immunomagnetic bead CD34 selection or FACS Aria ll sorted to obtain leukemic progenitors (LSC/CD34+CD38+Lin−). Malignant progenitors were then transplanted into neonatal RAG2−/−gc−/− mice, and 8 weeks post-transplant, mice were treated with SAR503, dasatinib and vehicle for 14 days. Following treatment, hematopoietic tissues were analyzed for human engraftment by FACS analysis.Our results revealed that single agent experiments with SAR503 had a cytostatic rather than a cytoreductive effect on BC LSC. The treatment alone (60 mg/kg twice daily administered by oral gavage) did not significantly reduce leukemic progenitor burden in the liver, spleen, bone marrow and peripheral blood. Conversely, combination therapy with SAR503 and dasatinib (50mg/kg/day) significantly reduced LSC progenitors in all tissues examined.Interestingly, we observed that dasatinib alone therapy reduced the LSC burden in the liver, spleen, and peripheral blood, but the bone marrow retained a significant population of BC LSC. Also we found that the GMP population, previously shown to be enriched for BC LSC (Jamieson et al NEJM 2004; Abrahamsson et al PNAS 2009), was preferentially localized in the bone marrow. As shown by our laboratory and others, LSC therapeutic resistance may be influenced by extrinsic cues provided by the niche (e.g. promoting quiescence). Because quiescence has been implicated in driving tyrosine kinase inhibitor resistance and LSC survival and because the bone marrow retains a resistant population, we decide to perform secondary transplantation experiments to determine relapse potential (self-renewal). LSC progenitors were isolated by immunomagnetic bead selection of human CD34+ cells from marrows and spleens of treated mice. After serially transplanting an equal number of this cells into secondary recipients, we observed a significant reduction in LSC serial transplantation only following combination treatment, suggesting that the combination therapy can abolish LSC self-renewal capacity and thereby potentially prevent relapse.To validate drug exposure, we have been performing both genomic and nanoproteomic analysis. Regarding the proteomics validation studies, we analyzed sorted LSC derived from spleen (pooled 5 mice per group) that were treated with vehicle or SAR503 for 14 days. The analysis was performed to detect status of p-JAK2, JAK2, p-STAT5 and B2-microglobulin (loading control). We observed a down regulation on the levels of p-JAk2 (active site Tyr 1007–08) and p-Stat5 (active site Tyr 694) (35% and 42% respectively), while no changes are observed for total JAK2 protein or B2M between both conditions.The full transcriptome sequencing, on sorted LSC treated with SAR503 alone and in combination with dasatinib, identified specific isoform changes in the JAK/STAT pathway that could be used as biomarkers of response and could explain the synergistic effect of the combination therapy. We have also characterized, at an isoform level, biomarkers of resistance that could explain relapse of disease after single agent therapy and we are currently validating these findings. Disclosures:No relevant conflicts of interest to declare.

Human Blast Crisis Leukemia Stem Cell Inhibition with a Novel Smoothened Antagonist.

AbstractAbstract 1223Sonic hedgehog (Shh) pathway activators, particularly smoothened (SMO), are implicated in driving mouse leukemia progression by promoting leukemia stem cell maintenance. Thus, we investigated the role of the Shh signaling pathway in a humanized mouse model of blast crisis (BC) CML as well as the effects of a novel SMO antagonist on leukemia stem cell (LSC) quiescence, survival and self-renewal both in vitro and in vivo. Methods:Human BC CML progenitor cells (n=3 patients) were treated for 7 days in vitro with vehicle (DMSO) or a clinical grade SMO antagonist (Pfizer; PF-04449913) and harvested for Shh target gene Q-PCR analysis. FACS-purified BC CML LSCs (CD34+CD38+Lin-PI-) were transplanted intrahepatically into newborn RAG2-/-gc-/- mice. When human progenitors were detectable in peripheral blood (8 weeks), mice were treated for 14 days daily by oral gavage with 200μl of drug or vehicle (50% PEG in HBSS). The treatment included dasatinib alone (50 mg/kg), PF-04449913 (100 mg/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, peripheral blood, spleen and liver and the number of myeloid sarcomas was evaluated. In addition, cell cycle analysis was also performed on the same hematopoietic tissues of vehicle compared with treated mice. To determine if self-renewal potential was abrogated by treatment, human leukemic progenitors were serially transplanted and myeloid sarcoma formation was assessed in secondary recipients after 8 weeks. Results:BC CML progenitors (n=3 patient samples) harbored increased expression of Gli and downregulation of Ptch1 transcripts compared to normal cord blood. Notably, Gli expression decreased following in vitro treatment with PF-04449913. Treatment with PF-04449913 alone in human BC CML LSC transplanted mice (n=12 exp., n=46 mice) significantly reduced spleen size (p ≤ 0.05). However, SMO inhibition alone did not significantly inhibit bone marrow engraftment compared with vehicle (n=12 exp., n= 47 mice). Although dasatinib alone decreases myeloid sarcoma formation (p ≤ 0.05), like the SMO antagonist alone it did not eradicate the LSC in the liver or bone marrow (n=12 exp., n=36 mice). Combination treatment (n=12 exp., n=34 mice) with PF-04449913 and dasatinib revealed a significant decrease in LSC hepatic engraftment (P≤0.05) compared with the PF-04449913 or dasatinib alone. Moreover, combination treatment completely eradicated the capacity of LSC to form myeloid sarcomas formation (0.0) in secondary transplant 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=16).Hematopoietic progenitor assays performed with normal cord blood CD34+ progenitors (n=3 samples) showed no reduction in colony survival following treatment with PF-04449913 (1μM) compared with vehicle. Furthermore, there was no significant inhibition of normal CD34+ colony replating following PF-04449913 compared with vehicle treatment. Cell cycle analysis demonstrated a reduction in quiescent human leukemic cells in the peripheral blood and marrow following SMO inhibitor treatment. Conclusions:Combination therapy with a potent BCR-ABL inhibitor, dasatinib, and a selective SMO inhibitor abrogates the self-renewal capacity of human imatinib resistant blast crisis leukemia stem cells. A phase I clinical trial with PF-04449913 is currently underway and a Phase 1b trial involving dasatinib and PF-04449913 for patients with advanced phase, imatinib resistant CML will begin in the near future. Disclosures:Jamieson:Pfizer: Research Funding; CIRM: Research Funding.