BACKGROUND This study evaluated the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of 8-chloro-adenosine (8-Cl-Ado) in patients with relapsed/refractory acute myeloid leukemia (AML). METHODS 8-Cl-Ado was administered daily for 5 days; the starting dose was 100 mg/m2 , the highest dose tested was 800 mg/m2 . The end points were toxicity, disease response, and PK/PD measurements. RESULTS The predominant nonhematologic toxicity was cardiac with grade ≥3 toxicity. Plasma PK in all patients suggested heterogeneity among patients, yet, some dose-dependency for the accumulation of 8-Cl-Ado. Two 8-Cl-Ado metabolites accumulated at similar levels to 8-Cl-Ado. Cellular PK in eight patients indicated accumulation of 8-Cl-ATP, which was associated with AML blast cytoreduction in peripheral blood. The authors determined the RP2D of 8-Cl-Ado to be 400 mg/m2 . CONCLUSIONS Given the cardiac adverse events observed, patients require monitoring for arrhythmias and QT interval during infusion. Although peripheral blood cytoreduction was observed, responses were transient, suggesting combination strategies will be required.

Supplemental Table 1. MCL cell line information.

Supplemental Table 3. Primary antibody information.

All four supplemental figure legends are in this file.

Supplemental Table 2. Patient diagnosis, leukocyte types and cytogenetics information

<p>Supplemental Figure 1. Effect of idelalisib on the cell cycle in MCL cell lines and primary cells. Supplemental Figure 2. Effect of idelalisib treatment on the AKT/mTOR-mediated translation regulation pathway in MCL cell lines. Supplemental Figure 3. Relationship of protein synthesis inhibition with growth inhibition and decrease of protein phosphorylation targets by idelalisib treatment in MCL lines and primary cells. Supplemental Figure 4. Composite immunoblot panels for MCL cell lines and primary cells to explain use of loading control in different figures.</p>

Covalent (c) Bruton's tyrosine kinase (BTK) inhibitors (BTKi) are effective agents for treatment of chronic lymphocytic leukemia (CLL). As single agent or in combination they result in long-term disease-free survival. However, some patients are intolerant to cBTKi while in others disease relapses due to pathogenic mutation in the C481 domain where cBTKi bind. More selective and noncovalent (nc) BTKi have proven to be efficacious for this patient population. Among ncBTKi, pirtobrutinib (previously known as Loxo-305) has been successfully used in the clinic (NCT03740529) for patients with CLL after cBTKi therapy. Previously treated patients with CLL entered on this protocol from Oct 2019 till Sept 2021 and received single agent oral pirtobrutinib until disease progression. Data from 27 CLL patients are available while one is lost to follow-up; baseline BTK status was wildtype (WT) in 10 and mutant (Mt) in 16 patients. Among these 26 patients, 13 (50%) progressed while on pirtobrutinib therapy (Table1). BTK profiling results at start of therapy show that among those who progressed, 11 patients had mutant BTK (85%) and 2 patients had WT-BTK (15%) suggesting that mutant BTK may be more likely to have disease recurrence while on pirtobrutinib therapy. Most common mutations were C418S, C418R, C481F, T4741 and PLCGy2 alone or in combination. Some patients with progression developed BTK clonal evolution with new nonC481 Mt-BTK clone or increase in variant allele frequency (VAF) of mt-BTK. BTK profiling at time of DP indicated that in some of these patients the original pathological clone of C481 Mt-BTK resolved. Disease progression (13 patients) typically occurred during the first two years of therapy with median time to progression 22 cycles for patients harboring WT-BTK and 14 cycles for those with Mt-BTK. Most of these patients (12) also showed high risk CLL features of TP53 mutation (9 patients), ATM/11qdel (9 patients), Notch1 mutation (4 patients), complex karyotype (6 patients) or trisomy 12 (3 patients). There are limited therapeutic options post pirtobrutinib progression. Therefore, we conducted pharmacological profiling for these patients by ex-vivo testing CLL cells post progression. We incubated peripheral blood mononuclear cells for three days with cBTKi - ibrutinib; glutathione inhibitor (also known as mtTP53 modulator) - APR-246; Bcl-2 antagonist - venetoclax; and Mcl-1 antagonist - AZD5991; as single agent and in double or triple combinations and apoptosis was assessed by flow cytometry. Endogenous cell death was negligible even after 3 days in suspension culture indicating highly resistant cell population. Single agent venetoclax at 50 nM was most effective with a median 70% cell death (range 40-90%) after 3 days. Interestingly, cells harboring mt-BTK appeared to be more sensitive to venetoclax (p = 0.01). APR-246 alone showed heterogeneity with 10% to 85% cell death yet it was able to sensitize cells to venetoclax, ibrutinib, and AZD5991 in both WT and mutant BTK patient cohorts. Loss of C481S original clone after pirtobrutinib therapy made cells sensitive to ibrutinib. Four (4) patients, both pretreatment and during therapy or progression samples were available. Intra-patient comparison demonstrated that compared to pretreatment sample, cells were sensitized to targeted agents after 10 - 14 cycles of pirtobrutinib therapy (Figure 1). Additional samples are being tested during this ongoing clinical trial. In conclusion, progression on pirtobrutinib therapy was more common in patients with Mt-BTK. Pharmacological profiling based on BTK status at disease progression showed venetoclax and APR-246 as the most effective single agents. Combination of APR-246 or venetoclax with ibrutinib or AZD5991 showed maximum cell death. Patients were re-sensitized to ibrutinib after loss of C481S clone. Genomic and genetic profile along with pharmacological landscape provide precision medicine for these heavily pretreated patients whose disease is resistant to BTK inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

It is known that 8-chloro-adenosine (8-Cl-Ado) is a novel RNA-directed nucleoside analog that targets leukemic stem cells (LSCs). In a phase I clinical trial with 8-Cl-Ado in patients with refractory or relapsed (R/R) AML, we observed encouraging but short-lived clinical responses, likely due to intrinsic mechanisms of LSC resistance. LSC homeostasis depends on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. We recently reported that 8-Cl-Ado and the BCL-2-selective inhibitor venetoclax (VEN) synergistically inhibit FAO and OXPHOS in LSCs, thereby suppressing acute myeloid leukemia (AML) growth in vitro and in vivo. Herein, we report that 8-Cl-Ado inhibits ribosomal RNA (rRNA) synthesis through the downregulation of transcription initiation factor TIF-IA that is associated with increasing levels of p53. Paradoxically, 8-Cl-Ado-induced p53 increased FAO and OXPHOS, thereby self-limiting the activity of 8-Cl-Ado on LSCs. Since VEN inhibits amino acid-driven OXPHOS, the addition of VEN significantly enhanced the activity of 8-Cl-Ado by counteracting the self-limiting effect of p53 on FAO and OXPHOS. Overall, our results indicate that VEN and 8-Cl-Ado can cooperate in targeting rRNA synthesis and OXPHOS and in decreasing the survival of the LSC-enriched cell population, suggesting the VEN/8-Cl-Ado regimen as a promising therapeutic approach for patients with R/R AML.

8-Chloro-adenosine (8-Cl-Ado) is currently in phase I clinical trial. Activation of p53 and transactivation of p21 regulate cell fate after genotoxic insult. Using HCT-116-isogenic-cell-lines, we evaluated the role of p53/p21 after 8-Cl-Ado-mediated response. Following 30 mu M 8-Cl-Ado treatment, RNA synthesis was inhibited, p53 protein was stabilized, and p21 expression was activated. None of the cell types were arrested in G1/S phase, however, cells lacking p53 were blocked in G2/M. These cells had the least increase in apoptotic cells, although clonogenic survival demonstrated equal inhibition in all 4 cell types. Collectively, irrespective of p53 and p21 status, 8-Cl-Ado-induced cytotoxicity was similar.

Pirtobrutinib (LOXO-305), a reversible inhibitor of Bruton’s tyrosine kinase (BTK), was designed as an alternative strategy to treat ibrutinib-resistant disease that develops due to C481 kinase domain mutations. The clinical activity of pirtobrutinib has been demonstrated in CLL, but the mechanism of action has not been investigated. We evaluated pirtobrutinib in 4 model systems: first, MEC-1, a CLL cell line overexpressing BTK WT , BTK C481S , or BTK C481R ; second, murine models driven by MEC-1 overexpressing BTK WT or BTK C481S ; third, in vitro incubations of primary CLL cells; and finally, CLL patients during pirtobrutinib therapy (NCT03740529, ClinicalTrials.gov). Pirtobrutinib inhibited BTK activation as well as downstream signaling in MEC-1 isogenic cells overexpressing BTK WT , BTK C481S , or BTK C481R . In mice, overall survival was short due to aggressive disease. Pirtobrutinib treatment for 2 weeks led to reduction of spleen and liver weight in BTK WT and BTK C481S cells, respectively. In vitro incubations of CLL cells harboring wild-type or mutant BTK had inhibition of the BCR pathway with either ibrutinib or pirtobrutinib treatment. Pirtobrutinib therapy resulted in inhibition of BTK phosphorylation and downstream signaling initially in all cases irrespective of their BTK profile, but these effects started to revert in cases with other BCR pathway mutations such as PLCG2 or PLEKHG5. Levels of CCL3 and CCL4 in plasma were marginally higher in patients with mutated BTK; however, there was a bimodal distribution. Both chemokines were decreased at early time points and mimicked BCR pathway protein changes. Collectively, these results demonstrate that pirtobrutinib is an effective BTK inhibitor for CLL harboring wild-type or mutant BTK as observed by changes in CCL3 and CCL4 biomarkers and suggest that alterations in BCR pathway signaling are the mechanism for its clinical effects. Long-term evaluation is needed for BTK gatekeeper residue variation along with pathologic kinase substitution or mutations in other proteins in the BCR pathway.

Abstract Background BCL‐2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Although VEN-containing regimens yield 60–70% clinical response rates, the vast majority of patients inevitably suffer disease relapse, likely because of the persistence of drug-resistant LSCs. We previously reported preclinical activity of the ribonucleoside analog 8-chloro-adenosine (8-Cl-Ado) against AML blast cells and LSCs. Moreover, our ongoing phase I clinical trial of 8-Cl-Ado in patients with refractory/relapsed AML demonstrates encouraging clinical benefit. Of note, LSCs uniquely depend on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. VEN inhibits OXPHOS in LSCs, which eventually may escape the antileukemic activity of this drug. FAO is activated in LSCs isolated from patients with relapsed AML. Methods Using AML cell lines and LSC-enriched blast cells from pre-treatment AML patients, we evaluated the effects of 8-Cl-Ado, VEN and the 8-Cl-Ado/VEN combination on fatty acid metabolism, glycolysis and OXPHOS using liquid scintillation counting, a Seahorse XF Analyzer and gene set enrichment analysis (GSEA). Western blotting was used to validate results from GSEA. HPLC was used to measure intracellular accumulation of 8-Cl-ATP, the cytotoxic metabolite of 8-Cl-Ado. To quantify drug synergy, we created combination index plots using CompuSyn software. The log-rank Kaplan–Meier survival test was used to compare the survival distributions of the different treatment groups in a xenograft mouse model of AML. Results We here report that VEN and 8-Cl-Ado synergistically inhibited in vitro growth of AML cells. Furthermore, immunodeficient mice engrafted with MV4-11-Luc AML cells and treated with the combination of VEN plus 8-Cl-Ado had a significantly longer survival than mice treated with either drugs alone (p ≤ 0.006). We show here that 8-Cl-Ado in the LSC-enriched population suppressed FAO by downregulating gene expression of proteins involved in this pathway and significantly inhibited the oxygen consumption rate (OCR), an indicator of OXPHOS. By combining 8-Cl-Ado with VEN, we observed complete inhibition of OCR, suggesting this drug combination cooperates in targeting OXPHOS and the metabolic homeostasis of AML cells. Conclusion Taken together, the results suggest that 8-Cl-Ado enhances the antileukemic activity of VEN and that this combination represents a promising therapeutic regimen for treatment of AML.

Although ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton's tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal-related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2-/-γc-/- mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non-BTK-targeted therapeutics.

To realize the full potential of promising new anticancer drugs, it is of paramount importance to administer them at the right dose. The aim of this educational article is to provide several opportunities to optimize anticancer drug dosing, focusing on oral targeted therapies. First, therapeutic drug monitoring can optimize exposure in individual patients, if the optimal concentration is known. This approach is of particular interest in regard to oral kinase inhibitors with high interindividual pharmacokinetic variability. If exposure is related to response, then therapeutic drug monitoring is potentially feasible, although the clinical utility of this approach has not yet been established. Other approaches to reduce variability include administration of more frequent, smaller doses and administration under optimal prandial conditions. However, for many drugs, the labeled dose has not been demonstrated to be the optimal dose; for such agents, the vast majority of patients may be receiving excessive doses, which results in excessive toxicity. Furthermore, administration of lower off-label doses may reduce both medical and financial toxicity. These strategies should be applied from registration studies to clinical practice, with the goal of better optimizing anticancer treatment.

Relapse of acute myeloid leukemia (AML) is attributed to the persistence of quiescent leukemia stem cells (LSCs). Bcl-2 inhibition has been shown to target primitive leukemia progenitors. Venetoclax (VEN) is a FDA-approved Bcl-2-selective inhibitor for the treatment of AML. Although the activity of single agent VEN in AML patients (pts.) is modest, clinical efficacy in newly diagnosed, older pts. unfit for intense chemotherapy has been shown when VEN is combined with the hypomethylating agents (HMAs) azacytidine and decitabine or with low-dose nucleoside analog cytarabine. We have recently shown that VEN in combination with HMAs augments oxidative stress in AML cells and provided a molecular mechanism for the VEN-HMA-regulated NF-E2-related factor 2 (Nrf2) antioxidant pathway that could explain the results observed in early clinical studies in AML. Although about 70% of pts. initially respond to these VEN treatment regimens, about 30% of pts. do not and diminished efficacy of VEN combination treatments have been observed in pts. harboring poor-prognosis markers such as FLT3-ITD. In addition, future relapse of a percentage of pts. treated with VEN combinations is expected. Thus, novel treatment options for are urgently needed. We previously reported that the ribose containing, RNA-directed nucleoside analog 8-chloro-adenosine (8-Cl-Ado) demonstrates cytotoxic activity against AML cells and LSCs in vitro and in vivo, without significantly affecting normal hematopoietic stem cells. Importantly, our initial, unpublished results from a phase I/II clinical trial with single agent 8-Cl-Ado in pts. with refractory/relapsed AML demonstrate encouraging clinical benefits. Moreover, we have reported that FLT3-ITD AML is particularly sensitive to 8-Cl-Ado, thus suggesting 8-Cl-Ado plus VEN as a potential novel therapeutic regimen for treatment of AML. We here report that the VEN plus 8-Cl-Ado combination inhibited in vitro growth and induced apoptosis in AML primary cells, LSCs and cell lines significantly more compared to treatment with the individual agents. For in vitro cell growth studies, combination indices of <1 for all experimental and calculated drug concentrations demonstrated strong synergy between the two drugs in 2 human AML cell lines (MV4-11 and KG-1a) and in AML cells isolated from 2 pts. Moreover, immune compromised NSG mice engrafted with FLT3-ITD MV4-11 cells survived significantly longer when treated with VEN (20 mg·kg‒1·day‒1, daily oral) plus 8-Cl-Ado (50 mg·kg‒1·day‒1; osmotic pump), as compared to single agent or vehicle-treated mice (p<0.006, VEN+8-Cl-Ado vs. 8-Cl-Ado; p<0.001 VEN+8-Cl-Ado vs. VEN). LSCs depend on amino acid metabolism-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for energy production. VEN is known to target LSCs through inhibition of OXPHOS by targeting amino acid uptake/metabolism. We report here that 8-Cl-Ado inhibited the FAO pathway and down-regulated the oxygen consumption rate (OCR), a marker for OXPHOS, in LSCs. However, whereas 500 nM of 8-Cl-Ado was sufficient to induce MV4-11 growth inhibition, 1 microM of 8-Cl-Ado was needed for maximum inhibitory effect on FAO. We also report that 8-Cl-Ado increased expression of the anti-apoptotic protein p53. It was previously reported that p53 induces FAO in LSCs. Knockdown of p53 by siRNA augmented the inhibitory effect of 8-Cl-Ado on FAO and OCR. Importantly, addition of VEN could completely overcome the p53-induced activation of FAO and OCR. Mechanistically, we show that 8-Cl-Ado inhibited ribosomal RNA (rRNA) synthesis, a prerequisite for cellular proliferation, through down-regulation of the transcription initiation factor 1 (TIF-IA) protein. Since TIF-1A negatively regulates p53 expression, the inhibition of TIF-1A by 8-Cl-Ado resulted in up-regulation of p53 and subsequent p53-induced upregulation of FAO and OCR, thus diminishing the suppressive effects of 8-Cl-Ado on FAO and OCR. We further show that the VEN plus 8-Cl-Ado combination strongly induced p53 signaling, as shown by activation and inhibition of downstream p21 and PCNA proteins, respectively. This combination also augmented DNA fragmentation and apoptosis in LSCs. Thus, our data suggest that the synergy seen in AML with the VEN plus 8-Cl-Ado combination can be explained at least in part due to augmented inhibition of FAO and OXPHOS and represents a promising novel treatment for AML. Disclosures Pullarkat: Dova: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marcucci:Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial). Rosen:Celgene: Speakers Bureau; NeoGenomics: Consultancy; Seattle Genetics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; Pebromene: Consultancy.

The first-in-class irreversible inhibitor of Bruton’s tyrosine kinase (BTK) ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL) [1]. Ibrutinib is indicated for all pati...

BCL-2 family proteins play a critical role in the survival of chronic lymphocytic leukemia (CLL) cells and a number of new agents have been developed to target pro-survival protein members of this ...

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD (+) MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.

Talimogene laherparepvec (T-VEC) has demonstrated efficacy for unresectable melanoma. We explored response patterns from a phase 2 study evaluating patients with unresectable stage IIIB–IVM1c malignant melanoma who received T-VEC plus ipilimumab or ipilimumab alone. Patients with objective response per modified irRC were evaluated for pseudo-progression (single ≥25% increase in tumour burden before response). Patients without pseudo-progression were classified by whether they responded within or after 6 months of treatment start; those with pseudo-progression were classified by whether pseudo-progression was due to increase in existing lesions or development of new lesions. Overall, 39% (n = 38/98) in the combination arm and 18% (n = 18/100) in the ipilimumab arm had an objective response. Eight responders (combination, n = 7 [18.4%]; ipilimumab, n = 1 [5.6%]) had pseudo-progression; most occurred by week 12 and were caused by an increase in existing lesions. These data reinforce use of T-VEC through initial progression when combined with checkpoint inhibitors. Trial Registration NCT01740297 (ClinicalTrials.gov; date of registration, December 4, 2012); 2012-000307-32 (ClinicalTrialsRegister.eu; date of registration, May 13, 2014).

AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values <1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016).