Larisa Balaian

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 inhibitory effect of anti-CD33 monoclonal antibodies on AML cell growth correlates with Syk and/or ZAP-70 expression

OBJECTIVES Acute myeloid leukemia (AML) cells express the cell surface antigen CD33 that can function as a downregulator of cell growth, mediating growth arrest and apoptosis. The protein kinase Syk is an essential element in several cascades coupling certain antigen receptors to cell responses. Recently we reported that CD33 recruits Syk for its signaling in AML cell lines. In this study, we further investigated the mechanism(s) of Syk engagement in CD33 signaling in primary AML samples. METHODS We investigated 25 primary AML samples for their proliferative response (3H-thymidine incorporation) and biochemical changes (Western blot analysis) to anti-CD33 mAb treatment. RESULTS Proliferation studies demonstrated that 14 (56%) of AML samples were responsive (R) while 11 (44%) were nonresponsive (n-R) to inhibitory antibody activity. Seven of 25 AML samples (28%) expressed undetectable levels of Syk. However, cells from two of these patients expressed the ZAP-70 protein kinase. In Syk/ZAP-70(+) samples, CD33 ligation inhibited proliferation in 70% of cases, while none of the Syk/ZAP-70(-) samples was responsive. There were significant biochemical differences between responder and nonresponder AML populations. In responder samples, CD33 ligation induced phosphorylation of CD33 andSyk and formation of the CD33/Syk complex. In nonresponder samples, CD33 was not phosphorylated, and Syk was in complex with the SHP-1 protein phosphatase constitutively. CONCLUSIONS Syk is an important component in the regulation of proliferation in AML cells. The differential response of AML cells to CD33 ligation is associated with the level of the Syk expression.

ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis

Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling.

Direct effect of bispecific anti-CD33×anti-CD64 antibody on proliferation and signaling in myeloid cells

Bispecific anti-CD33 x anti-CD64 antibody (BsAb) directly inhibited proliferation and colony formation of human acute myeloid leukemia cell lines, without affecting the function of normal monocytes. Addition of BsAb to normal monocytes induced tyrosine phosphorylation of Cbl and Vav, association of these molecules with CD33, and downstream signaling. In leukemia cells that were insensitive to BsAb treatment, Vav and Cbl were constitutively phosphorylated and, therefore, constitutively associated with CD33. Direct growth inhibition is an additional mechanism by which BsAb may be useful in the therapy of AML.

A Phase I/II Trial of the Combination of Azacitidine and Gemtuzumab Ozogamicin for Treatment of Relapsed Acute Myeloid Leukemia

Micro‐Abstract In view of the single‐agent activity of azacitidine and gemtuzumab ozogamicin (GO) in patients with relapsed acute myeloid leukemia, we studied a combination strategy in a phase I/II trial to potentially further improve outcomes. GO/azacitidine therapy was relatively well tolerated and resulted in response rates similar to those with GO used as monotherapy at higher doses. Introduction Treatment with hypomethylating agent therapy might enhance anti‐CD33 monoclonal antibody‐mediated cytotoxicity against acute myeloid leukemia (AML) blasts through epigenetic effects on Syk and SHP‐1 expression. Patients and Methods In the present phase I/II study, we treated patients with relapsed or refractory AML with azacitidine, followed by 2 doses of gemtuzumab ozogamicin (GO) at 6 mg/m2, the Food and Drug Administration‐approved dose and schedule at study initiation. We sought to determine the maximum tolerated dose and clinical activity of this combination therapy. Secondarily, we aimed to determine whether baseline Syk and SHP‐1 expression can be used as predictive biomarkers of treatment response. Results The established maximum tolerated dose was azacitidine 75 mg/m2 daily for 6 consecutive days, followed by GO 6 mg/m2 on days 7 and 21. Of the 50 evaluable patients, 12 (24%) obtained complete remission (CR) or CR with incomplete peripheral blood recovery (CRp). No dose‐limiting toxicities were observed in phase I, and no patient developed hepatic sinusoidal obstructive syndrome. Although no significant correlation was found between Syk and SHP‐1 expression and the clinical response to combination therapy, in vitro studies repeatedly demonstrated that azacitidine‐treated AML cells had an increased response to GO treatment. Conclusion Our study found that the combination of GO with azacitidine is relatively well tolerated, with response rates similar to those with GO monotherapy at higher doses. Differences in the GO drug schedule, dose level, and frequency might explain the discrepant response rates between our study and others, suggesting that the optimal GO dose remains unclear, especially when combined with hypomethylating agent therapy.

Abstract 3338: Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche

Introduction Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche. Methods Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n = 5), MF (n = 5) and relapsed AML (n = 4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n = 4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib. Results Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated >50% inhibition, in another 40% it was 20-50% and in 20% inhibition was Conclusions Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia. Citation Format: Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche. [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 3338.

Abstract 4865: Differential survival and self-renewal of LSC and HSC in aged and leukemic niches

Background: Acute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Evidence indicates that in the development of AML deregulation of stem cells activity is as important as deregulation of the microenvironment. We have recently reported that inflammatory changes in the aging bone marrow (BM) niche via aberrant RNA editing and splicing may predispose patients to leukemic transformation. However, little is known about the functional effects of niche driven aberrant RNA processing in the AML pathogenesis, as well as of role of aberrant RNA editing and splicing in human aged stem cell fate, dormancy and regeneration in the context of microenvironment. Here we investigate: 1/ the LSC impacts to the BM niche and 2/ changes in the activity of BM niche cells contribute to AML pathogenesis. Methods: BM CD34- cell from normal donors and AML patients were used for the development of the primary human stromal monolayers : young (y-BM, n=3); old (a-BM, n=3) and AML (n=3). Human CD34+ cells were selected from AML primary samples (n=6). As a control, CD34+ cells from cord blood (CB, n=5), y-BM (n=3) or a-BM (n=3) were used for the co-culture experiments and then plated in survival and self-renewal assays. Results: AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood HSC (n=5) lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. Aged BM also impaired survival and self-renewal of cord blood HSC (n=3) in stromal co-culture models. In similar experiments HSC from a-BM (n=3) demonstrated significantly higher survival and self-renewal capacity then co-cultured with y-BM stroma (n=3) compared to a-BM stroma (n=3). Conditioned media (CM) both from-, or co-culture with-, old or AML stroma impaired HSC survival and self-renewal. Notably, co-culture conditions resulted in a greater reduction in survival and self- renewal capacity, suggesting that cell-cell contact or unstable secreted factors exacerbate the effects. Cord blood HSC were also co-cultured with AML-derived stroma or CM , and this severely impaired their survival and self-renewal. Moreover, pre-treatment of the HS-5 cells with CM from a-BM or AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood HSC (n=3) survival and self-renewal. Conclusions: Together these data indicate that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic and aged stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of AML. Citation Format: Larisa Balaian, Leslie Crews, Anna Kulidjian, Edward Ball, Catriona Jamieson. Differential survival and self-renewal of LSC and HSC in aged and leukemic niches. [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 4865. doi:10.1158/1538-7445.AM2014-4865

Abstract 219: Splicing inhibitors reduce human AML CD34+ cell survival and self-renewal during MDS/AML evolution in a leukemia stem cell supportive niche assay.

Introduction. Myelodysplastic syndromes (MDS) are relatively common neoplasms of hematopoietic stem cells, which commonly evolve to acute myeloid leukemia (AML). Recent results suggest that MDS evolution is controlled by mutations in splicing related genes and epigenetic modifiers of gene expression. Alternative splicing driven by these mutations has been implicated in the evolution of MDS to AML. However, little is known about the cell type and context specific functional effects of these mutations on leukemia stem cells (LSC) that promote AML therapeutic resistance. Therefore, we investigated 1) the effect of splicing inhibitors on LSC survival and self-renewal during the MSD/AML progression in a bone-marrow stromal co-cultures that recapitulates key aspects of the human LSC niche and 2) the genomic mutations in LSC and non-LSC populations during disease progression. Methods. Whole exome sequencing on CD34+ and CD34- cells before and after MDS/AML progression . Mouse bone marrow cell lines transfected to produce human SCF, IL3 and G-CSF, were used as a stromal monolayer. Human CD34+ cells were selected from MDS and AML primary samples (n=6). As a normal control, cord blood CD34+ cells (CB, n=3) were utilized. Two SF3B1-targeted splicing inhibitors: FD 895 and a FD-analog were added at the initiation of co-culture at concentrations ranging from 0.1 to10 uM. Results. Whole exome DNA sequencing analysis revealed that a loss of chr21 was observed at different frequencies in CD34- and CD34+ cells. We identified a RUNX1 missense mutation with increasing prevalence in CD34+ during progression. Notably, loss of heterozygosity and a missense mutation in the histone methyltransferase EZH2 gene, implicated in MDS progression, was detected only in CD34+ post-progression to AML. After 2 weeks of stromal co-culture, survival of the cells was investigated by colony forming assay in methylcellulose. While the splicing inhibitors demonstrated no cytotoxicity towards normal CB, MDS and AML samples showed a dose-dependent inhibition of colony formation. To analyze the effect of splicing inhibitors on LSC self-renewal, replating assays were performed. While compounds at high doses mediated only a slight decrease in colony formation in CB samples, MDS and AML samples exhibited a dose dependent inhibition of LSC survival (38.2+/-8.1% p Citation Format: Larisa Balaian, Michael Burkart, Shawn Yost, Sophie Rozenhak, Edward D. Ball, Kelly Frazer, Olivier Harismendy, Catriona Jamieson. Splicing inhibitors reduce human AML CD34+ cell survival and self-renewal during MDS/AML evolution in a leukemia stem cell supportive niche assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 219. doi:10.1158/1538-7445.AM2013-219