ALBERT

Description: Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8–10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation–related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Description: 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.

Description: Background Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established. Methods Whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013). Results We performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance. Conclusion In summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. Disclosures Jamieson: Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Description: Introduction Leukemia stem cells (LSC) in chronic myeloid leukemia (CML) are important in disease progression and relapse. Conventional therapy with tyrosine kinase inhibitors (TKIs), although very effective at reducing bulk CML cells, frequently fail to eliminate LSC residing in the protective bone marrow niche. Thus, there is an unmet need for combination therapy that simultaneously targets bulk disease and eliminates LSC in order to prevent disease progression and relapse. Stem cell niches are often rich in hyaluronic acid. CD44, the main receptor for hyaluronic acid, and some of its splice variants are frequently overexpressed on cancer stem cells, including LSC. In this study we aimed to evaluate the in vivo anti-LSC activity of CD44 monoclonal antibody (mAb) RG7356. Methods and Results For anti-CD44 LSC inhibition studies, immunodeficient RAG2-/-γc-/- neonatal mice were intrahepatically transplanted with human BC LSC from imatinib responsive and resistant patients. Once engraftment was verified, mice were treated with control IgG1 (30mg/kg/week), dasatinib (25mg/kg/day), CD44 mAb (RG7356) (30mg/kg/week) or a combination of CD44 mAb (RG7356) and dasatinib. The mice were sacrificed after two weeks of therapy and bone marrow, spleen, peripheral blood and myeloid sarcomas were analyzed by FACS to assess CML LSC burden. In the imatinib responsive patient, CD44 mAb single agent therapy showed an 85% reduction of LSC in bone marrow, 98% reduction in spleen and 91% reduction in peripheral blood. CD44 mAb therapy thus showed an efficacy equal to that of dasatinib, which showed an 87%, 98% and 98% reduction in bone marrow, spleen and peripheral blood respectively. Combination therapy (CD44 mAb + dasatinib) completely eradicated any trace of CML LSC in all tissues analyzed. In the imatinib resistant patient, dasatinib very effectively reduced the number of myeloid sarcomas and blasts but did not inhibit LSC survival in bone marrow. In contrast, CD44 mAb single agent therapy effectively reduced the number of CML LSC to 51% in bone marrow, 96% in spleen and 93% in peripheral blood. Conclusions This study demonstrates that a human CD44 specific mAb, RG7356, significantly reduces CML LSC survival. Notably, TKI resistance of CML LSC, can be overcome by treatment with a human CD44 specific mAb, RG7356, as it sensitizes CML LSC residing in malignant niches to dasatinib. From these results, RG7356 appears to be an excellent antibody for future combination clinical studies aimed at eradicating CML. Disclosures: Runza: employee of Roche Diagnostics GmbH: Employment. Weigand:Roche Diagnostics GmbH: Employment. Jamieson:J&J: Research Funding; Sanofi: Consultancy; Roche: Research Funding.

Description: Introduction Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are generated from progenitors that have aberrantly activated self-renewal pathways thereby resulting in tyrosine kinase inhibitor (TKI) resistance. The telomerase complex, consisting of a reverse transcriptase subunit (TERT), an RNA template subunit (TERC), and a protective shelterin scaffold, transcriptionally modulates the Wnt/b-catenin self-renewal pathway. Many malignancies, including BCR-ABL TKI resistant blast crisis CML (BC CML), exhibit robust telomerase activity thereby prompting the development of imetelstat, a competitive inhibitor of telomerase enzymatic activity. Imetelstat is a covalently lipidated 13-mer oligonucleotide that binds with high affinity to the TERC subunit. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms. However, the role of imetelstat in selective self-renewing LSC inhibition in CML had not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized LSC primagraft studies to investigate the capacity of imetelstat to selectively inhibit LSC self-renewal and to determine the mechanism of action. Methods and Results Cytoscape analysis of RNA-seq data derived from FACS-purified progenitors from human blast crisis (BC; n=9) compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples revealed transcriptional upregulation of b-catenin, LEF1, TCF7L1, ABL1 and other key genes within the TERT interactome suggesting a role for TERT activation in human BC LSC generation. Human progenitor LSC-supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 mM significantly inhibited (p

Description: Introduction Malignant 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 Results CD44 Isoform Expression Analysis To 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 Overexpression To 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 LSC Humanized 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. Conclusions Upregulation 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. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 910 In blast crisis transformation of CML (BC CML), the leukemia stem cells (LSC), via the acquisition of both enhanced survival and self-renewal capacity, become increasingly resistant to BCR-ABL targeted tyrosine kinase inhibition and thus often contribute to relapse after treatment, pointing to the need for alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. Janus kinase 2 (JAK2) plays an important role in BCR–ABL + cell survival and has profound effects on self-renewal and lineage commitment of normal and leukemic hematopoietic stem cells, through the activation of the transcription factor signal transducer and activator of transcription 5 (STAT5). To determine if JAK/STAT signaling pathway activation is related to CML progression, LSC from human Chronic Phase (CP CML) and BC CML samples were sorted using FACS Aria (Lin-CD34+CD38+) and analyzed using splice-isoform specific q-RT-PCR. Our results showed that, compared to CP CML, BC LSC harbor enhanced mRNA expression of BCR-ABL, JAK2 and STAT5A isoforms, confirming that progression of CP to BC, in CML LSC, is marked by activation of JAK/STAT pathway. Therefore, we investigated the response of BC CML LSC to a clinical grade JAK2 inhibitor, SAR302503 (Sanofi, Cambridge, MA) alone or in combination with a potent BCR-ABL inhibitor, dasatinib, in vivo. After two weeks of treatment, RAG2−/−gc−/− mice intrahepatic transplanted with BC LSC, showed a significant (p