ALBERT

Description: Binding of CD33 on AML cells by monoclonal antibodies (mAb) mediates cytotoxicicity on AML cells modulated by the protein tyrosine kinase Syk and the phosphatase SHP-1. We have demonstrated that Syk- negative AML cells are relatively resistant to the effects CD33 ligation, but after exposure to the hypomethylating agent 5-azacytidine (5-aza) they become sensitive to the effects of both unconjugated and chemically-conjugated (gemtuzumab ozogamycin, GO) anti-CD33 mAb. Here we tested a panel of 40 primary AML samples for the expression of SHP-1. In 13% of the cases SHP-1 was undetectable. Anti-CD33 mAb and GO induced growth inhibition more effectively in AML cells that expressed SHP-1. Among SHP-1-positive samples, 69% demonstrated significant growth inhibition in response to CD33 ligation. In contrast, none of the SHP-1-negative samples responded to anti-CD33 mAb. These results show a correlation between SHP-1 expression and responsiveness of AML cells to CD33 ligation. However, 5-aza treatment restored SHP-1 expression and, therefore, increased the anti-proliferative effects of anti-CD33 mAb and GO. In 40% of SHP-1-negative samples, the AML cells were only marginally inhibited by 5-aza or anti-CD33 mAb alone, whereas the combination produced a more than additive effect in AML cells where 5-aza induced re-expression of SHP-1. The effect of GO was more than doubled by 5-aza in these cells. Total inhibition of DNA synthesis in the presence of 5-aza plus anti-CD33 mAb or GO reached 60–70% and was similar in SHP-1-positive and SHP-1-negative cells. Moreover, 5-aza significantly enhanced of GO-mediated cytotoxicity in AML progenitor cells. In a NOD/scid mouse model, which permits growth of human AML cells and allows measurement of in vivo therapeutic effects of therapeutic strategies for AML, we tested whether combined treatment of the mice with 5-aza would enhance the cytotoxicity of anti-CD33 mAb and GO. Suboptimal doses of 5-aza by itself, as well as treatment with murine anti-CD33 mAb alone did not cause significant cytoreduction. However, combined treatment of mice with anti-CD33 mAb and 5-aza, resulted in a significant response. Treatment with GO mediated up to 60% inhibition of AML cell proliferation. Combined treatment of mice with GO and 5-aza resulted in reduction of leukemia cells by 〉80%. These data show an interaction of 5-aza and anti-CD33/GO in an in vivo AML model. Based on these data, we hypothesize that the combination of 5-aza and GO may be a potent therapy for patients with AML. Moreover, Syk and SHP-1 may serve as biomarkers of leukemia cell response. Therefore, we initiated a clinical trial of 5-aza and GO combined therapy. Six patients with relapsed AML have been treated in a dose escalation of 5-aza preceeding GO (6 mg/m2 times two). All 6 were Syk-positive, while SHP-1 expression was detected in 4 samples and absent in two. 2 days of 5-aza treatment in vivo induced re-expression of SHP-1 in both previously SHP-1 negative patient cells. Moreover, significant increases were observed in the levels of Syk protein in one baseline positive sample and 1 SHP-baseline positive sample. Study of the effects of 5-aza alone ex vivo on the baseline patient cells showed no significant effect on leukemia cell proliferation. However, importantly, in all 6 samples 5-aza more than doubled the AML cells’ response to cytotoxic effects of GO and naked anti-CD33 mAb. These results suggest that 5-aza may augment the effects of anti-CD33 mAb therapy through demethylation of SYK, SHP-1, and possibly other genes. The clinical efficacy of the combined therapy requires further study.

Description: Introduction Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. Secondary AML (sAML) arises from prior myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN), and occurs in patients with an average age of 〉65. Because of the typically advanced age of this population, patients currently have few effective treatment options available after leukemic transformation. We and others have recently identified a key role for enzymatic RNA editing activity in cancer progression, and in particular in leukemia stem cell (LSC) generation. In hematopoietic stem and progenitor cells, adenosine deaminase acting on dsRNA-1 (ADAR1) is the most abundantly expressed RNA editing gene. However, the role of abnormal RNA editing activity has not been elucidated in healthy human bone marrow aging and age-related MDS with a high risk of transforming to sAML. Therefore, we established whole transcriptome-based RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, which provides novel RNA-based functionally relevant biomarkers of aging, MDS and progression to sAML. Methods Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin- HSC) and progenitor cells (CD34+CD38+Lin- HPC) from aged (average age = 65.9 y/o) versus young (average age = 25.8 y/o) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 y/o) and MDS (average age = 63.8 y/o). Comparative gene set enrichment analyses (GSEA) and RNA editing profiles were identified for normal and malignant progenitor cell aging. Results Aberrant RNA editing activity has recently been shown to be induced in multiple cancers, and has been implicated as a malignant reprogramming factor. Comparative whole transcriptome RNA sequencing (RNA-seq) and single nucleotide variant analyses revealed widespread increases in RNA editing rates in aged versus young HPC, and in human sAML LSC compared with age-matched normal progenitors. Moreover, RNA editing rates, represented as adenosine (A) to inosine/guanosine (G) changes at known RNA editing loci, were increased in sAML compared with MDS progenitors. The differential expression of certain sites is as high as 70%, which can be readily detected by RESS-qPCR. These data suggest that during aging niche-dependent RNA editing deregulation contributes to MDS progression to sAML. Interestingly, the highly edited loci in sAML LSC were distinct from loci that were differentially edited in aged versus young HPC, suggesting that pro-inflammatory conditions in sAML may trigger RNA editing of a unique set of transcripts, including predominantly RNA processing-related gene products and transcription factors. Notably, several loci in transcripts of APOBEC3C/D that we previously found were associated with blast crisis transformation of chronic myeloid leukemia also displayed enhanced editing in sAML LSC, but not aged versus young HPC. Conclusions Detection of aberrant RNA processing provides novel biomarkers as well as potential therapeutic targets for sAML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders. We have identified commonly RNA-edited transcripts in multiple hematologic malignancies, which could be developed clinically and as companion diagnostic targets for LSC-targeted therapeutics. Disclosures Jamieson: CTI Biopharma: Research Funding; Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Description: Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

Description: Abstract 2735 Leukemia stem cells (LSC) play a crucial role in the development and progression of chronic myeloid leukemia (CML). Although BCR-ABL targeted tyrosine kinase inhibitors (TKI), such as dasatinib, can eradicate the majority of CML cells, they frequently fail to eliminate the dormant, niche-resident LSC that are hypothesized to drive CML relapse. Cumulative evidence from CML cell lines and CD34+ primary patient cells suggests that increased expression of pro-survival BCL2 family members contributes to TKI resistance and CML progression. However there is a relative dearth of data on BCL2 family expression in primary CML LSC and on the role of these proteins in TKI resistance in selective niches. Full transcriptome RNA sequencing revealed that LSC switch from pro-apoptotic to pro-survival BCL2 family member splice isoform expression during progression from chronic phase to blast crisis CML. Using splice isoform-specific qRT-PCR, we identified overrepresentation of long (pro-survival) compared with short (pro-apoptotic) MCL1, BCLX, and BCL2 isoforms in blast crisis LSC compared with chronic phase and normal progenitors. Following intrahepatic transplantation of blast crisis LSC into neonatal RAG2−/−gc−/− mice, LSC engrafted in the marrow niche were quiescent, were dasatinib resistant and upregulated BCL2 expression. These data led us to speculate that inhibition of BCL2 in dasatinib-resistant LSC may sensitize LSC to TKI therapy. Treatment with a high-potency, novel pan-BCL2 family inhibitor, sabutoclax, in vitro led to a dose-dependent increase in apoptosis along with a decrease in the frequency of leukemic progenitors compared to vehicle treated controls. Normal human cord blood progenitor cells were less sensitive to sabutoclax treatment with IC50 approximately five times higher than that for blast crisis CML cells (210 nM versus 43 nM). Moreover, sabutoclax treatment did not inhibit cord blood colony formation or colony replating in vitro. Treatment of CML LSC-transplanted mice with sabutoclax led to a significant reduction in LSC burden in all hematopoietic organs analyzed. Sabutoclax treatment in vivo also sensitized surviving bone marrow blast crisis LSC to dasatinib treatment ex vivo. Importantly, there was no reduction in normal progenitor engraftment in bone marrow following sabutoclax treatment. These results demonstrate that marrow niche blast crisis CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan, BCL2 antagonist, sabutoclax, at doses that spare normal hematopoietic progenitors. While BCL2 splice isform switching promotes LSC survival and TKI resistance, pan-BCL2 family member inhibition with sabutoclax eliminates LSC and may form the cornerstone of a clinical strategy to avert cancer progression and relapse. Disclosures: No relevant conflicts of interest to declare.

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: Abstract 3776 Cumulative 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.