Potential Approaches Versus Approved or Developing Chronic Myeloid Leukemia Therapy

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, continued use of these inhibitors has contributed to the increase in clinical resistance and the persistence of resistant leukemic stem cells (LSCs). So, there is an urgent need to introduce additional targeted and selective therapies to eradicate quiescent LSCs, and to avoid the relapse and disease progression. Here, we focused on emerging BCR-ABL targeted and non-BCR-ABL targeted drugs employed in clinical trials and on alternative CML treatments, including antioxidants, oncolytic virus, engineered exosomes, and natural products obtained from marine organisms that could pave the way for new therapeutic approaches for CML patients.

Contribution of BCR-ABL molecular variants and leukemic stem cells in response and resistance to tyrosine kinase inhibitors: a review

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm generated by reciprocal chromosomal translocation, t (9; 22) (q34; q11) in the transformed hematopoietic stem cell. Tyrosine kinase inhibitors (TKIs) target the mature proliferating BCR-ABL cells, the major CML driver, and increase overall and disease-free survival. However, mutant clones, pre-existing or due to therapy, develop resistance against TKIs. BCR-ABL1 oncoprotein activates various molecular pathways including the RAS/RAF/MEK/ERK pathway, JAK2/STAT pathway, and PI3K/AKT/mTOR pathway. Stimulation of these pathways in TKI resistant CML patients, make them a new target. Moreover, a small proportion of CML cells, leukemic stem cells (LSCs), persist during the TKI therapy and sustain the disease in the patient. Engraftment of LSCs in the bone marrow niche and dysregulation of miRNA participate greatly in the TKI resistance. Current efforts are needed for determining the reason behind TKI resistance, identification, and elimination of CML LSC might be of great need for cancer cure.

Leukemic Stem Cells of Monocytic AMLs Are Not-Resistant to BCL-2 Inhibition

Treatment with Hypomethylating agents (HMA) such as 5-Azazytidine (AZA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows results superior to treatment with AZA alone (DiNardo et al., 2020, NEJM). However upfront resistance and relapse following initial response remain major obstacles. It has recently been proposed that monocytic differentiation predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). This appears to be due to increased expression of other anti-apoptotic proteins such as MCL-1 in monocytic AMLs, which conveys resistance to AZA/VEN therapy, as survival of leukemic cells in these patients is no longer dependent on BCL-2. However, an independent study found no impaired outcome in patients with monocytic AMLs treated with HMA/VEN (Maiti et al., 2020, Blood, ASH abstract). Here, we show that monocytic AML cell lines and bulk cells of monocytic primary AML cells are indeed intrinsically resistant to AZA/VEN treatment. However, in a collective of 30 patients treated with HMA/VEN at Heidelberg University Medical Center between 2018 and 2020, monocytic differentiation assessed by flow cytometry was not an independent risk factor for refractory disease. We hypothesized that the conflicting data may be caused by intra-patient heterogeneity of AZA/VEN sensivitity and assessed killing efficiency in various immunophenotypic subpopulations of 12 primary AML patient samples in vitro. The CD64 +CD11b +, differentiated blast population made up >50% of leukemic cells in monocytic and <20% in primitive samples and showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain leukemic stem cells (LSC). In contrast, we found immature CD64 -CD11b - GPR56 + LSC to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive types of primary AMLs. As expected, LSCs from either monocytic or primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential for the effect of AML therapy. Next, we investigated expression of BCL-2, MCL-1 and BCL-xL in the same primary patient samples and observed high MCL-1 expression in monocytic AML samples. However, MCL-1 expression was restricted to the CD64 +CD11b + population whereas in the LSC sub-populations robust expression of BCL-2 but low levels of MCL-1 and BCL-xL were detected, independent of whether monocytic or primitive AMLs were analyzed. To further validate the sensitivity of LSCs of monocytic AML to BCL-2-I, we established a platform combining BH-3 profiling with multi-color flow cytometry, allowing for single cell assessment of cellular dependencies on independent apoptotic pathways. We found that LSCs of both AML types show high VEN/BAD but low MS-1 induced apoptosis, functionally confirming the expression patterns of BCL-2 and MCL-1. As LSCs are rare in monocytic samples, investigation of samples in bulk are dominated by MCL-1 expressing and resistant non-LSCs, explaining the overall higher MCL-1 expression/survival of monocytic compared to immature AML cells. However, our data uncovers sensitivity of LSCs to AZA/VEN independent of overall monocytic or primitive sample classification and provide a mechanistic explanation for the clinical data of Maiti et al. and our Heidelberg AML collective, which found no increased resistance of monocytic AMLs to AZA/VEN treatment. Disclosures Unglaub: JazzPharma: Consultancy, Other: travel costs/ conference fee; Novartis: Consultancy, Other: travel costs/ conference fee. Schlenk: Abbvie: Honoraria; Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding.

High-Throughput Chemical Screen on Acute Myeloid Leukemia Stem Cells Identifies Novel Anti-LSC Compounds

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence. High-throughput screens of human AML LSCs are not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. We were able to optimize conditions for a 4-week in vitro large-scale expansion (>600 million bulk) of the primary human AML sample 8227 (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). These optimized conditions enabled the isolation and maintenance of the LSC-containing fraction for a chemical screen. We isolated the CD34+ LSC-containing fraction (>90% purity) and performed a high-throughput screen of 11,166 chemical molecules using a CellTiter Glo assay followed by a counter screen against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells. From this HT screen, a total of 61 hits had >70% inhibition on CD34+ 8227 cells and <30% inhibition on CD34+ CB cells. We also identified glucocorticoids, which were also identified in our prior small-scale anti-LSC screen where they were found to specifically drive human LSCs to terminally differentiate (Laverdière & Boileau, et al., 2018). We then performed dose response assays for each candidate compounds and confirmed 35 potent anti-LSC compounds with IC 50 < 1 μM. This refined the types of compounds to including anti-apoptotic inhibitors, GSK inhibitors, protease inhibitors, metabolism inhibitors, HDAC inhibitors, BET inhibitors, nucleic acid synthesis inhibitors, cell cycle inhibitors and Wnt/β-catenin inhibitors. This is interesting as some of the classes of these compounds (inhibitors of GSK, BET, nucleic acid synthesis, Wnt/β-catenin and metabolism) have been shown to target bulk and leukemic stem cells in AML in vitro and in vivo. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs confirmed through in vitro and in vivo assays. Our goal is to be able to understand and establish the molecular mechanisms and biomarkers on primary functional LSCs. Disclosures No relevant conflicts of interest to declare.

Flow Cytometry Based Identification of CD26+ Leukemic Stem Cells in the Peripheral Blood Has a Potential Role in the Rapid Diagnosis of Chronic Myeloid Leukemia

Background: Demonstration of t(9;22)(BCR-ABL1) fusion is gold standard for the diagnosis of chronic myeloid leukemia (CML). We performed a flowcytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for the diagnosis of CML and its utility for detection of residual disease in CML patients on therapy. Methods: Patients of CML/ CML on follow-up were included and peripheral (PB) and/or bone marrow (BM) samples were utilized for flowcytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. Under 'lyse-wash-stain-wash' sequence, the sample was incubated with a pre-titrated custom-made antibody cocktail in a 'test' tube containing CD45, CD34, CD38 and CD26 mo-abs. Acquisition was carried out on BD FACS Canto II and analysis was done with Diva Software. Clinical data including demographic details, complete blood count and BM findings were also noted. Results: A total of 104 samples (63 PB and 41 BM) from 64 patients [confirmed & treatment naïve CML (n=30), CML on follow-up (n=15), non-CML (n=19)] were tested. The median (range) time for reporting of PB/BM examination, molecular genetic studies and flow cytometry for CD26+ CML LSCs was 5 (3-11 days), 4 (3-6 days) and 1 (0-1 day) respectively. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (Median=0.07%, range 0.002%-26.79%), and also in 8/15 patients of the follow-up group, who also reported persisting levels of BCR-ABL1. None of the patients in the non-CML group and follow-up CML patients with negative RT-PCR results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r=0.917). Conclusion: Flow cytometric assessment for CD26+ LSCs is quick with reporting time of even less than an hour. Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is independent of BCR-ABL1 transcript type and its role in residual disease monitoring needs further investigation. Disclosures No relevant conflicts of interest to declare.

CD123-Targeted Nano-Curcumin Molecule Enhances Cytotoxic Efficacy in Leukemic Stem Cells

Acute myeloblastic leukemia (AML) is a disease with a high rate of relapse and drug resistance due to the remaining leukemic stem cells (LSCs). Therefore, LSCs are specific targets for the treatment of leukemia. CD123 is specifically expressed on LSCs and performs as a specific marker. Curcumin is the main active compound of a natural product with low toxicity for humans. It has been reported to inhibit leukemic cell growth. However, curcumin is practically insoluble in water and has low bioavailability. In this study, we aimed to formulate curcumin nanoparticles and conjugate with the anti-CD123 to overcome the low water solubility and improve the targeting of LSCs. The cytotoxicity of both curcumin-loaded PLGA/poloxamer nanoparticles (Cur-NPs) and anti-CD123-curcumin-loaded PLGA/poloxamer nanoparticles (anti-CD123-Cur-NPs) were examined in KG-1a cells. The results showed that Cur-NPs and Cur-NPs-CD123 exhibited cytotoxic effects on KG-1a cells with the IC50 values of 74.20 ± 6.71 and 41.45 ± 5.49 µM, respectively. Moreover, anti-CD123-Cur-NPs induced higher apoptosis than Cur-NPs. The higher uptake of anti-CD123-Cur-NPs in KG-1a cells was confirmed by using flow cytometry. In conclusion, the anti-CD123-Cur-NPs formulation improved curcumin’s bioavailability and specific targeting of LSCs, suggesting that it is a promising drug delivery system for improving the therapeutic efficacy against AML.

Low Exposure Extended Dosing Mimicking Clinical Exposures of the Oral Formulation of Azacitidine Results in a Sustained Hypomethylation and Targets Leukemic Stem Cells

Acute Myeloid Leukemia (AML) is characterized by uncontrolled proliferation of incompletely differentiated myeloid stem/ progenitor cells. Despite recent advances in therapy, high rates of clinical relapse, even in patients who achieve complete remission, remains a critically unmet need. One of the strategies to prolong remission post-induction in AML is to employ effective maintenance therapies. Oral Azacitidine (Oral-AZA; CC-486) is the first and only currently approved maintenance therapy in AML. However, the mechanism of action of Oral-AZA and whether it is differentiated from the Injectable-AZA used in AML induction therapy, remains unclear. In this work, we explore the mechanism of Oral-AZA by in vitro modelling of the relative clinical exposures of Oral-AZA vs Injectable-AZA in AML cell line models. Following Vu et al (Nat. Commun. 2020), we used the Injectable-AZA concentration (1 μM aZA) as a single dose (HELD - High Exposure Limited Duration). A fractionated dose of 0.2 μM each day over 5 days (LEED - Low Exposure Extended Duration) was used to model Oral-AZA. Azacitidine incorporates into RNA and DNA of AML cells leading to hypomethylation driven gene expression changes. We found that HELD but not LEED dosing produced an acute anti-proliferative effect in sensitive AML cell lines suggesting potential for a non-hypomethylation mediated/integrated stress response (ISR) driven mechanism. This ISR effect was rescued by co-treatment with ISRIB, an ISR inhibitor. With HELD, we observed robust ATF4 activation as early as 6 hours that was sustained up to 24 hours. In contrast, LEED induced modest and transient ATF4 activation. Thus, an Injectable-AZA-like regimen activated the ISR pathway more robustly than an Oral-AZA-like regimen. Interestingly, decitabine, a DNA-only incorporating cytidine analog did not activate ISR. In AML cell lines, the target of Azacitidine, DNMT1 was depleted (about 90% compared to control) within 24 hours in both HELD and LEED regimens. However, LEED produced a more sustained DNMT1 loss, up to 7 days whereas in HELD, DNMT1 protein levels recovered 96 hours post-dosing. Given the relative level and duration of DNMT1 loss, we hypothesized that LEED would lead to a more durable hypomethylation. We performed whole genome bisulfite sequencing (WGBS) in 3 AML cell lines (OCI-AML2, MV-4-11 and SKM1) at 48- and 96-hours post-initiation of HELD and LEED dosing. Consistent with the DNMT1 depletion kinetics, at 48 hours we observed almost 75% global DNA hypomethylation with both HELD and LEED treatments. At 96 hours, HELD demonstrated a recovery effect and reverted to 50% hypomethylation. In contrast, LEED showed up to 85% hypomethylated sites, demonstrating the durability of the hypomethylation mediated by an Oral-AZA-like (LEED) regimen. Next, we explored the effect of Oral-AZA-like dosing in the differentiation of leukemic stem cells (LSCs) towards a more mature phenotype. In an in vitro LSC model (OCI-AML-20) with flow cytometry analysis, LEED dosing resulted in a greater depletion (2-fold more) of LSCs (CD34+/38- or 38 low) and concomitant enrichment of cells with more differentiated phenotype (CD34+/38+) compared to HELD dosing. To further validate these observations at the transcriptomic level, we performed single cell RNAseq in OCI-AML-20 cells at different timepoints (3, 5 and 7 days). Data were analyzed using a classifier to identify leukemic myeloid cell lineages (Van Galen et al., Cell 2019). Compared to untreated cells, at day 7, both LEED and HELD dosing resulted in an increase of GMP and promonocytes and those differences were more pronounced with Oral-AZA-like (50% GMP and 20% promonocytic) than the Injectable-AZA-like regimen (28% GMP and 12.5% promonocytic). Taken together, our in vitro modeling of the clinically relevant exposures of Oral-AZA vs Injectable-AZA by using HELD and LEED dosing strategies show a shift of the molecular mechanism between the clinical entities. Our work demonstrates that an Injectable-AZA-like regimen mediates cytotoxicity through an early ISR driven effect. Oral-AZA mechanism leads to a more sustained pharmacodynamic DNA hypomethylation effect that in turn could be linked to a differentiation inducing effect on the LSC population. Disclosures Jeyaraju: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Alapa: Bristol Myers Squibb: Current Employment. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Risueño: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Wang: Bristol Myers Squibb: Current Employment. Subramanyam: Bristol Myers Squibb: Current Employment. Sriganesh: Bristol Myers Squibb: Current Employment. Anand: Bristol Myers Squibb: Current Employment. Jain: Bristol Myers Squibb: Current Employment. Reddy: Bristol Myers Squibb: Current Employment. Ghosh: Bristol Myers Squibb: Current Employment. Kyriakopoulos: Bristol Myers Squibb: Current Employment. Lailler: Rancho Biosciences: Current Employment. Hartl: Rancho Biosciences: Current Employment. Lopes de Menezes: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Hagner: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.