Malignant A-to-I RNA editing by ADAR1 drives T cell acute lymphoblastic leukemia relapse via attenuating dsRNA sensing.

Leukemia-initiating cells (LICs) are regarded as the origin of leukemia relapse and therapeutic resistance. Identifying direct stemness determinants that fuel LIC self-renewal is critical for developing targeted approaches. Here, we show that the RNA-editing enzyme ADAR1 is a crucial stemness factor that promotes LIC self-renewal by attenuating aberrant double-stranded RNA (dsRNA) sensing. Elevated adenosine-to-inosine editing is a common attribute of relapsed T cell acute lymphoblastic leukemia (T-ALL) regardless of molecular subtype. Consequently, knockdown of ADAR1 severely inhibits LIC self-renewal capacity and prolongs survival in T-ALL patient-derived xenograft models. Mechanistically, ADAR1 directs hyper-editing of immunogenic dsRNA to avoid detection by the innate immune sensor melanoma differentiation-associated protein 5 (MDA5). Moreover, we uncover that the cell-intrinsic level of MDA5 dictates the dependency on the ADAR1-MDA5 axis in T-ALL. Collectively, our results show that ADAR1 functions as a self-renewal factor that limits the sensing of endogenous dsRNA. Thus, targeting ADAR1 presents an effective therapeutic strategy for eliminating T-ALL LICs.

Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells.

Pediatric acute myeloid leukemia (pAML) is typified by high relapse rates and a relative paucity of somatic DNA mutations. Although seminal studies show that splicing factor mutations and mis-splicing fuel therapy-resistant leukemia stem cell (LSC) generation in adults, splicing deregulation has not been extensively studied in pAML. Herein, we describe single-cell proteogenomics analyses, transcriptome-wide analyses of FACS-purified hematopoietic stem and progenitor cells followed by differential splicing analyses, dual-fluorescence lentiviral splicing reporter assays, and the potential of a selective splicing modulator, Rebecsinib, in pAML. Using these methods, we discover transcriptomic splicing deregulation typified by differential exon usage. In addition, we discover downregulation of splicing regulator RBFOX2 and CD47 splice isoform upregulation. Importantly, splicing deregulation in pAML induces a therapeutic vulnerability to Rebecsinib in survival, self-renewal, and lentiviral splicing reporter assays. Taken together, the detection and targeting of splicing deregulation represent a potentially clinically tractable strategy for pAML therapy.

Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.

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

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.

RNA Splicing Modulation Selectively Impairs Leukemia Stem Cell Maintenance in Secondary Human AML.

Age-related human hematopoietic stem cell (HSC) exhaustion and myeloid-lineage skewing promote oncogenic transformation of hematopoietic progenitor cells into therapy-resistant leukemia stem cells (LSCs) in secondary acute myeloid leukemia (AML). While acquisition of clonal DNA mutations has been linked to increased rates of secondary AML for individuals older than 60 years, the contribution of RNA processing alterations to human hematopoietic stem and progenitor aging and LSC generation remains unclear. Comprehensive RNA sequencing and splice-isoform-specific PCR uncovered characteristic RNA splice isoform expression patterns that distinguished normal young and aged human stem and progenitor cells (HSPCs) from malignant myelodysplastic syndrome (MDS) and AML progenitors. In splicing reporter assays and pre-clinical patient-derived AML models, treatment with a pharmacologic splicing modulator, 17S-FD-895, reversed pro-survival splice isoform switching and significantly impaired LSC maintenance. Therapeutic splicing modulation, together with monitoring splice isoform biomarkers of healthy HSPC aging versus LSC generation, may be employed safely and effectively to prevent relapse, the leading cause of leukemia-related mortality.

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 ADAR1's 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.

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.

Anti-CD33 monoclonal antibodies enhance the cytotoxic effects of cytosine arabinoside and idarubicin on acute myeloid leukemia cells through similarities in their signaling pathways.

OBJECTIVE: Chemotherapy agents (CA) such as cytosine arabinoside (ara-C), idarubicin (IDA), and etoposide (VP-16) are widely used in the treatment of acute myeloid leukemia (AML) However, their effects on signaling pathways leading to cytotoxicity have only been described recently. Ligation of the leukemia-associated antigen CD33 by anti-CD33 monoclonal antibody (mAb) also results in signaling events that induce a downregulation of cell growth. We examined the possibility that anti-CD33 mAb and CA might cooperate in mediation of growth inhibition in primary AML samples and AML cell lines. MATERIALS AND METHODS: We investigated two AML cells lines and 14 primary AML samples for their proliferative response ((3)H-thymidine incorporation), colony formation, and biochemical (Western blot analysis) to anti-CD33 mAb treatment combined with chemotherapy agents. RESULTS: CD33 ligation induced a significant increase in ara-C- or IDA- but not VP-16-or Bryostatin-mediated inhibition of proliferation and colony formation. Ara-C and IDA induced SHP-1 and SHP-2 protein tyrosine phosphatase (PTPs) phosphorylation and Lyn/SHP-1 complex formation, while VP-16 and Bryostatin did not. CD33 ligation, however, mediated phosphorylation of these PTPs and Syk/SHP-1 complex formations. Combined treatment of AML cells by ara-C or IDA with anti-CD33 mAb resulted in higher levels of SHP-1 phosphorylation. Reduction in SHP-1 by short interfering RNA abrogated these effects. CONCLUSION: These data suggest that combined incubation of leukemia cells with anti-CD33 mAb and ara-C or IDA, but not VP-16 or Bryostatin, independently triggers similar events in the downstream signaling cascade, and therefore leads to additive antiproliferative effects and enhanced cytotoxicity.

Inhibition of acute myeloid leukemia cell growth by mono-specific and bi-specific anti-CD33 x anti-CD64 antibodies.

Bi-specific anti-CD33 x anti-CD64 antibodies (BsAb) mediated more potent and longer-lasting inhibition of proliferation of human leukemia cell lines and primary acute myeloid leukemia (AML) samples compared to mono-specific anti-CD33 mAb. There were no differences between these two antibodies in cellular internalization over time. The inhibitory effect of BsAb was mimicked by a mouse IgG2a subclass mono-specific anti-CD33 mAb. These findings indicate that enhanced inhibition of proliferation was caused by simultaneous ligation of both CD33 and CD64 molecules. We conclude that inhibition of leukemia cell growth initiated by BsAb during prolonged exposure may have therapeutic value for the treatment of AML.

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.