Leukemic Stem Cells Research Articles

Cladribine and Low-Dose Cytarabine-Based Salvage Therapy for Relapsed/Refractory AML in a Predominantly Venetoclax-Exposed Cohort

Introduction: Relapsed/refractory acute myeloid leukemia (R/R AML) remains challenging to treat. Unfit patients receive either targeted therapy for actionable mutations or low-intensity chemotherapy, often a hypomethylating agent with venetoclax (HMA/ven). Aggressive regimens, such as a purine analog with high-dose cytarabine, are reserved for fit patients. A regimen using similar drug classes, comprised of cladribine (clad), low-dose cytarabine (LDAC), and venetoclax (clad/LDAC/ven), has demonstrated favorable efficacy and safety for frontline AML treatment in older patients, according to recent phase 2 studies [Kadia 2018 Lancet Hematology; Kadia 2022 Journal of Clinical Oncology]. Interestingly, monocytic differentiation is a proposed mechanism of venetoclax resistance, and monocytic leukemic stem cells rely on purine metabolism, thus suggesting unique cladribine sensitivity [Pei 2023 Cancer Discovery]. The utility of clad/LDAC with or without venetoclax in R/R AML is not well characterized, especially in today's evolving treatment landscape. Methods: In this single-center retrospective cohort study, we report efficacy and safety outcomes of a salvage regimen using a clad/LDAC backbone in adult patients with R/R AML between 2019 and 2023. For induction, patients received cladribine 5 mg/m2 IV daily days 1 to 5 and cytarabine 20 mg/m2 IV daily days 1 to 10. A third agent, such as venetoclax at a target dose of 400 mg oral daily days 1 to 21, was added at the provider's discretion. Dose reductions were allowed based on performance status and organ function. Re-induction was considered if the bone marrow biopsy demonstrated inadequate response. Patients who responded received clad/LDAC-based consolidation or proceeded to allogeneic hematopoietic cell transplant (alloHCT). Results: A total of 35 patients received clad/LDAC-based salvage induction for R/R AML. Median age was 68 years old, and patients had a median of 1 prior line of therapy. Half of patients had secondary AML, and most had adverse risk disease. The most common regimen received was clad/LDAC (49%), followed by clad/LDAC/ven (43%). Among 32 patients evaluable for response, the overall response rate (ORR) was 38%, with complete response (CR) of 16% and complete response with incomplete count recovery (CRi) of 22%. The median progression-free survival (mPFS) was 1.4 months (range 0.4-22.8), and median overall survival (mOS) was 3.4 months (range 0.4-22.8). At 6 months, PFS was 11% and OS was 26%. At 12 months, PFS was 11% and OS was 15%. For responders, 33% achieved negative measurable residual disease (MRD), 33% proceeded to alloHCT, mPFS was 3.3 months (range 0.9-22.8), and mOS was 6.4 months (0.9-22.8). Mutational profiling of responders revealed ASXL1, CSF3R, NF1, SETBP1, and U2AF1 to be most the common. In a subgroup of 21 patients who had progressive or refractory disease with frontline HMA/ven, clad/LDAC-based salvage resulted in ORR 37%, CR 16%, CRi 21%, MRD negativity 57% among responders, mPFS 1.4 months (range 0.4-20.9), and mOS 2.3 months (range 0.4-21.8). Clad/LDAC-based induction was well tolerated, with no cases of treatment-related mortality. Common adverse effects included febrile neutropenia (74%), grade 1 or 2 elevated bilirubin (49%), grade 1 transaminitis (48%), rash (43%), nausea/vomiting (37%), and grade 1 acute kidney injury (31%). One third of patients received clad/LDAC-based regimen in the outpatient setting. Conclusions: Clad/LDAC and clad/LDAC/ven represent feasible salvage induction strategies for R/R AML that may be administered outpatient and associated with minimal toxicity. Favorable response rates occurred in a subgroup of patients who failed frontline HMA/ven, in which there is an unmet need.

Targeting Venetoclax Resistance in TP53-Mutated Acute Myeloid Leukemia

Introduction: The combination therapy of Venetoclax (Ven) and Azacitidine (Aza) has shown remarkable improvements in the treatment of acute myeloid leukemia (AML) patients not eligible for chemotherapy. Nevertheless, patients with TP53-mutated AML are more prone to have a refractory disease or develop rapid resistance. A recent study indicated that a combined expression ratio of BCL-2, BCL-XL and MCL-1 proteins in leukemic stem cells predicts clinical response to Ven+Aza (Waclawiczek et al. 2023). Furthermore, our previous work has demonstrated that erythroid and megakaryoblastic leukemia blasts are more dependent on BCL-XL than BCL-2 (Kuusanmäki et al. 2023). Emerging evidence suggests that the erythroid-biased differentiation and gene signature is enhanced in many TP53-mutated patients (Rodriquez-Meira et al. 2022). Here we hypothesized that in TP53 -mutated AML, leukemic blasts correspond more closely to megakaryocyte-erythroid progenitors (MEPs) leading to an unfavorable BCL-2 family expression ratio, which can be associated with poor responses to Ven. Consequently, we aimed to identify novel compounds targeting this specific patient subgroup. Methods: We utilized flow cytometry to analyze the mean fluorescent intensity (MFI) of BCL-2 family proteins (BCL-2, BCL-XL, MCL-1) in the CD34+ blast cells of 16 bone marrow-derived AML samples taken before Ven+Aza therapy ( TP53mut n=8, TP53wt n=8). Ex vivo drug testing was performed with eight compounds and blast-specific drug responses were analyzed after 48 hours with flow cytometry. RNA sequencing was performed on CD34+ or CD117+ cells enriched from 35 pre-Ven+Aza treatment samples. To assess erythroid signature scores, we calculated the mean expression of six different erythroid genes ( GATA1, KLF1, ZFPM1, GATA2, GYPA, TFRC) for each patient, following the approach by Rodriquez-Meira et al. 2022. The expression levels of 19 intracellular proteins, including BCL-2, BCL-XL, MCL-1, were quantified from the corresponding cell population of the samples by mass cytometry (CyTOF). Results: In the comparison of BCL-2 family protein expression and treatment responses, BCL-XL levels were significantly higher (p=0.03) in the treatment-resistant group, while no differences were found in BCL-2 or MCL-1 ( Figure 1A). In the TP53-mutated blasts, a trend of decreased BCL-2 was identified compared to the TP53 wild-typeblasts (p=0.08), but no difference in BCL-XL or MCL-1 levels was noted. However, all three Ven+Aza-refractory TP53-mutants exhibited high BCL-XL expression compared to the treatment-responsive TP53-mutated patients. Next, we assessed whether an erythroid-like phenotype was associated with TP53 mutation status by computing the erythroid gene signature scores from the RNAseq data of enriched blasts. Erythroid scores were increased among TP53-mutated samples (p=0.04), and interestingly, a higher erythroid score was linked to lower BCL-2 protein expression, regardless of TP53 mutation status ( Figure 1B). Finally, we assessed the drug efficacy of eight pre-selected compounds, including BCL-2 family inhibitors. As expected, Ven was ineffective in the TP53-mutated samples. In contrast, the TP53-mutated blasts showed higher ex vivo sensitivity to Navitoclax (BCL-2/BCL-XL inhibitor) (Nav IC50=90nM, Ven IC50>1000nM). Additionally, LCL-161 (IAPs inhibitor) demonstrated notable efficacy in the TP53-mutants and was significantly more effective compared to the wild-type controls (LCL-161 IC50=300nM, Ven IC50>1000nM). Discussion: Here, we show that increased BCL-XL protein expression is associated with clinical resistance to Ven+Aza. Furthermore, the link between erythroid gene signature and BCL-2 expression suggests that especially those AML blasts harboring TP53 mutations might have impaired BCL-2 expression due to a closer resemblance to MEPs. The increased sensitivity of TP53-mutated AML cells to LCL-161 demonstrated in the study, highlights the potential of targeting extrinsic apoptosis pathway via inhibiting the inhibitors of apoptosis proteins (IAPs). Furthermore, co-targeting of BCL-2/BCL-XL might provide alternative therapeutic approach in Ven resistant TP53-mutated patients. These findings are currently being validated with a larger patient cohort.

A DNA Methylation Signature Predicts Clinical Outcome in Pediatric AML Patients

Pediatric Acute Myeloid Leukemia (AML) is a heterogenous disease with a dismal outcome. Recently, transcriptomic-based leukemic stemness scores have highlighted the importance of DNA methyltransferase 3B in pediatric AML prognosis, which invites a deeper investigation of leukemic stem cells through the scope of their DNA methylome. Other studies have shown the prognostic impact of genome-wide methylation burden (PMID: 29928480, 33080932). As the World Health Organization has recognized methylation analysis as the most apt technology for determining lineage and cell population origins of tumors (PMID: 34921008), it is befitting to evaluate the DNA methylome as a prognostic tool in hematopoietic malignancies. Thus, we hypothesize that a DNA methylation-based signature has the potential to enhance the prognostic precision in pediatric AML. In this study, we applied machine learning approaches to DNA methylation data from 924 pediatric AML patients (only including data from samples with diagnostic bone marrow specimens) from three Children's Oncology Group clinical trials: AAML1031, AAML0531, and AAML03P1, available through GSE190931, GSE124413 and GDC_TARGET-AML, respectively. Clinical endpoints available were defined as :i) Minimal residual disease after induction 1 (MRD1): positive MRD1 if ≥ 1 leukemic cell per 1,000 mononuclear bone marrow cells (≥ 0.1%) determined by flow cytometry; ii) event-free survival (EFS) defined as the time from study enrollment to induction failure, relapse, secondary malignancy, death, or study withdrawal for any reason, with event-free patients censored on last follow-up; iii) overall survival (OS) defined as the time from study enrollment to death, with living patients censored on the date of last follow-up. Processing of the raw data followed best practices in the literature using SeSAMe (PMID: 30085201, 27924034). To independently validate the findings derived from the discovery cohort, we processed in parallel meDNA array data from bone marrow specimens at diagnosis from 201 AML patients treated on the multi-site clinical trials AML02 and AML08 (NCT00136084 and NCT00703820). As step 1, we performed Epigenome-Wide Association Study with Cox Proportional Hazards Regression (Cox-PH EWAS) to identify CpGs with methylation levels most predictive of OS, controlling for risk group categories. At p-value threshold of 1.0e-6, 167 CpGs were associated with OS, which were chosen as candidate CpGs for step 2 analysis using LASSO-based Cox-PH model, with 1,000 iterations of 10-fold cross-validation. 35 CpGs were consistently selected as non-zero coefficients in at least 95% of the models and were used to define the CpG based signature. Patients were further categorized into either Low MethylScoreAML Px (n=563, 60%) or High MethylScoreAML Px (n=361, 40%) groups using recursive partitioning statistics. In the discovery cohort, patients with High MethylScoreAML Px demonstrated a markedly poorer probability of OS (HR 3.91, p<0.0001) and EFS (HR 2.61, <0.0001) in comparison to the low MethylScore. These results were further tested in the validation cohort (n=201) for OS (HR 2.45, p=0.0006) and EFS (HR 2.15, p=0.0004). Notably, in subgroup analyses for both discovery and validation, MethylScoreAML Px remained statistically significant in multivariable models for EFS and OS with adjustments for MRD1 status, risk group, FLT3 status, white blood cell count at diagnosis, and patient age, indicating it as a robust, independent model supplementing currently known prognostic factors. In conclusion, this study unveils MethylScoreAML Px, a methylation-based prognostic risk score that robustly predicts clinical outcome in pediatric AML patients. To our knowledge, we are the first group to describe a Cox-PH-EWAS analysis in pediatric AML, as well as the first to devise and validate a prognostic risk score in >1000 patients from multiple pediatric-only trials. Future directions of research are focused on testing MethylScore in fresh peripheral blood samples using long read, real-time Oxford Nanopore sequencing technology, and comparing it with other risk scores.

Uncovering the Transcriptional Landscape of CML Development and Therapeutic Interventions Via State-Transition Modeling

Chronic Myeloid Leukemia (CML) is driven by expression of the BCR-ABL fusion gene. Despite the success of treatment with tyrosine kinase inhibitors (TKIs), the disease-originating leukemic stem cells frequently persist through therapy, and disease cannot be eradicated. Thus, new biomarkers beyond BCR/ABL may be necessary to assess disease state and treatment response, and to identify novel targets and therapeutics that could lead to a cure. Herein, we used time-series RNA-seq to molecularly characterize the transcriptional states of CML and predict disease evolution and treatment response. Using weekly blood draws from a murine tetracycline (Tet)-inducible BCR/ABL expression model of chronic phase CML and non-BCR/ABL expressing controls ( Fig 1A), we applied state-transition theory to mathematically model CML development. We constructed a CML state-space using dimensionality reduction to the full transcriptomic data of the time-series CML and control mice. From the sample distribution in the state-space, we empirically derived healthy (no BCR/ABL) and perturbed (BCR/ABL-induced) potentials ( Fig 1B). Using these potentials, our model described the transcriptome evolution during CML development as a particle undergoing Brownian motion. We used the Fokker-Planck (FP) solution of the Langevin equation of motion to both accurately model the transcriptome and predict the future dynamics from a single sample used as an initial condition. While control mice remain in the region of the state-space corresponding to health, each of the BCR/ABL expressing mice moved toward CML state as early as one week after BCR/ABL induction and before detection of BCR/ABL mRNA expression. Of note, no significant difference was observed between the experimental and the FP predicted trajectories and survivals, supporting the accuracy of our approach in predicting disease evolution and outcome at the earliest time points. The CML potential was described as a three well potential with three stable (Early c1, Transition c3, Late c5) and two unstable (Early Transition c2 and Late Transition c4). Differentially expressed genes (DEGs) corresponding to Early c1, Transition c3, Late c5 states were identified by comparing with control mice. Importantly, the state-space model also allowed assessment of whether each DEG had a pro- or anti-CML effect. At the Early state c1, we identified 78 DEGs; 55% of them were anti-CML, indicating an early attempt of the biological system to prevent disease development. By GSEA, we showed that the anti-CML Hallmark gene sets included EMT, angiogenesis, hypoxia and the only pro-CML gene set was IL2/Stat5. In contrast, at the Transition c3 and Late c5 states, we identify 366 and 1858 DEGs, respectively; nearly all these DEGs had a pro-CML effect and were enriched for Hallmark gene sets involving inflammation, angiogenesis and metabolism. Using CML state-space as a pseudotime ordering of the CML samples, we also studied modules of non-linear gene expression dynamics. This approach revealed changes in gene expression rates (inflection points) coinciding with the unstable critical points (c2 and c4) that separated the otherwise stable states. The genes in these transition modules included immune/inflammation processes and likely represented those genes that drove CML state-transition. To investigate the evolution of disease “in reverse” (i.e., after the CML phenotype developed), we treated two cohorts of CML mice, one with a TKI (nilotinib) for 4 weeks and one with Tet to turn off the expression of BCR/ABL (Tet-off, Tet-on [TOTO]). We adapted the state-transition model to a “treatment state-transition” model by introducing a specific parameter, g, which both accurately described and predicted individual treatment efficacy. We showed that although the circulating leukemic cells were suppressed by TKI, the disease state recovered only from c5 to c3, but never reached the health (control) state, and the treated mice rapidly relapsed once treatment was completed. Conversely, the TOTO cohort, which had the BCR/ABL expression completed turn off, returned stably to the healthy state. In conclusion, our state-transition model provides a theory-based analytic framework for investigating disease dynamics and predicting treatment response and outcome. Importantly, can be translated into the clinic for novel transcriptomic-based diagnostic approaches.

Iadademstat and Gilteritinib for the Treatment of FLT3-Mutated Relapsed/Refractory Acute Myeloid Leukemia: The Frida Study

Background: Frontline and relapsed /refractory (R/R) acute myeloid leukemia (AML) patients carrying fms-related tyrosine kinase 3 [FLT3]-mutations (FLT3 mut+) benefit from FLT3 inhibitors (FLT3i) that have emerged as active therapeutic agents for this subset of AML encompassing up to 30-40% of all AML cases. The FLT3i gilteritinib, as monotherapy for R/R AML pts, has improved outcomes but the duration of remission achieved is transient and often brief. The Phase 3 randomized ADMIRAL trial of gilteritinib demonstrated a 20% CR rate and 2.8 months event-free-survival (EFS) (Perl, et al., NEJM 2019). The development of new targeted agents which synergize with FLT3 inhibitors, and ideally also inhibit the emergence of resistance, is therefore a major medical need in AML. Iadademstat (iada/ORY-1001) is a specific, oral, potent, covalent inhibitor of the epigenetic Lysine-Specific Demethylase 1 (LSD1/KDMA1) enzyme. Epigenetic dysregulation is a hallmark of AML. Up to 70% of recurring mutations in AML patients target epigenetic regulators of gene expression, resulting in myeloid differentiation blockade and enhanced leukemic stem cell renewal, underscoring the potential of epigenetic therapies to change the natural history of the disease. Preclinically, iada produces striking synergy with FLT3is, particularly gilteritinib in FLT3 wild-type and FLT3 mut+ AML cells and in derived cell lines resistant to venetoclax, azacitidine and other FLT3is (Sacilotto et al., 2022 Eur J. of Cancer 174S1). The novel MOAs generating this synergy in combinations include activation of a pro-differentiating epigenetic transcriptional program with simultaneous suppression of MYC-driven target genes (Yashar, et al. 2023, Mol. Cancer Res). The recently completed Phase 2 study of iada in combination with azacitidine (ALICE) produced a high rate of composite remission, including complete remission (CR) and CR with incomplete count recovery (CRi) (mostly MRD negative), as well as durable responses, in treatment naïve, unfit AML patients (pts), without exacerbating the toxicity profile of azacitidine (Salamero et al., 2022, Blood S1). The FRIDA study (NCT05546580) aims to establish the safety, tolerability, and the recommended phase 2 dose (RP2D) of the combination of iada plus gilteritinib in FLT3 mut+ R/R AML. Methods: Adult pts with body weight ≥50 Kg and ECOG 0-2, with FLT3 mut+ R/R AML, after no more than 2 prior lines of therapy, are enrolling. Certain patients with prior exposure to FLT3is including gilteritinib may be eligible. Up to 18 pts, in a 3+3 escalation phase, will receive iada at doses of 75 to 150 ug, orally, in 5 days ON - 2 days OFF schedule, with continuous gilteritinib, at 120 mg/day orally. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s (determined based on all available data from escalation pts including PK, target engagement (TE), safety, tolerability, and emerging activity) will be enrolled. Primary endpoints of the study are safety and RP2D determination, based on the same 5 criteria outlined above. Bayesian posterior probability efficacy monitoring will be performed periodically for each dose cohort in the expansion phase. Bayesian efficacy futility and early stopping boundary will be applied during the monitoring. Posterior probability criterion (Prob (CR>0.3) ≥ 0.60) at the end of the study will warrant additional development. The safety of the combination treatment will also be continuously evaluated during expansion following a Bayesian design stopping rule.Secondary endpoints include overall survival, EFS, CR, CR/CR with partial hematologic recovery (CRh), overall response rates, time to response, duration of response, and transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis. At the time of the submission, FRIDA is enrolling a second dose level cohort in escalation phase and plans to have the 15 sites open to accrual in the US by the end of 2023. Additional sites will be added for a subsequent randomized controlled double-blinded FRIDA 2 study to assess the efficacy of the iada and gilteritinib combination in R/R FLT3 mut+ AML.

Single Cell Analysis of Genes Involved in Asymmetric Hematopoietic Stem Cell (HSC) Division in Chronic Myeloid Leukemia (CML) : Evidence of the Regulation of Tetraspanin CD63 during Leukemia Progression

Tyrosine kinase inhibitor (TKI) therapies have profoundly changed the natural history and survival of CML patients. However, the majority of the patients remain on long-lasting therapies and switch one TKI to another, either because of intolerance or resistance. All TKI cessation trials showed approximately 50% of leukemia recurrence rates even after several years of TKI therapies, suggesting the persistence of residual leukemic stem cells in patients in deep molecular response (DMR). The resistance and persistence of CML HSC are related to intrinsic (BCR-ABL independent survival) or extrinsic (leukemic niche ) mechanisms some of which have been extensively studied. However, there has been no data with regard to the role of asymmetric cell division pathways in the persistence and progression of CML stem cell clone. Indeed, During asymmetric division process of hematopoietic stem cells, some proteins are differently segregated between daughter HSCs such as endosome associated tetraspanin proteins (CD53, CD63) and mitochondria-associated fission regulator Dynamin related protein-1(DRP1). Similarly, it has been shown that asymmetric organelle inheritance can influence HSC decisions towards differentiation versus self-renewal. Interestingly, the tetraspanin CD63 is known to be involved in the quiescence of human and murine HSC via activation of TGF-beta pathway, CD63 hi cells exhibiting increased self-renewal potential after the asymmetric division with reduced differentiation potential. Materials and Methods We have analyzed the transcriptome performed in a cohort of CML patients in chronic phase (CP) and blast phase (BP) (GSE4170) as well as in a validation cohort of RNA-sequencing (GSE100026). Similarly, single cell transcriptome analysis of Lin- CD34+ CD38- CML progenitors (GSE76312) were included in the study evaluating the expression of genes involved in asymmetric cell division. We have then generated an asymmetric hematopoietic stem cell risk score using a panel of genes involved in the asymmetric division of HSCs and correlated these results with the transcriptome of the CML cohorts above. A pseudotime analysis was performed to evaluate single cell trajectories of the genes identified. Results To determine the potential role of asymmetric cell division markers in the progression of CML, we have analyzed the expression of several of these genes during CML progression in two independent transcriptome cohorts. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules which were found to be down-regulated during blast crisis as compared to chronic phase. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules down regulated during blast crisis as compared to chronic phase suggesting less of asymmetric properties during advanced phase of the disease. The down regulation in patients in blast crisis was confirmed at single cell level for the expression of SELL, CD63, NUMB, HK2 and LAMP2. CD63 expression has also been found to be of interest in CML chronic phase. Indeed, single cell neural network and deep learning in CP samples showed that the CD63 expression was the best asymmetric gene marker predicting a poor prognosis and this appeared to be induced by opposite regulation of HK2 and NUMB. In patients in CP-CML, the single cell trajectory reconstitution revealed the major regulatory role of CD63 highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, PDIA3, all genes known to be involved also in hematopoietic malignancies such as myelodysplasia, AML and ALL. Conclusion We show here for the first time the identification of the expression of genes involved in the asymmetric division of CML HSC. Among these genes we identified the tetraspanin CD63 as being down-regulated during CML progression. The expression of the genes involved in the asymmetric HSC division should require further studies in CML, either to predict persistence of CML or progressive disease. Figure 1 : Single cell transcriptome of Lin- CD34+ CD38- CML progenitors in CP links CD63 expression to progressive disease. A/ Piechart of counts for chronic phase cells analyzed according to prognosis; B/ pseudotime transformation of single cell transcriptome according patient prognosis; C/ Pseudotime expression heatmap; D/ Pseudotime expression of CD63 in CP.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

VISTA a Potential New Immuno-Oncology Target in Acute Myeloid Leukemia

V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family member highly expressed on circulating and intra-tumoral myeloid cells. VISTA is a negative checkpoint protein that inhibits anti-tumor T cell responses. In multiple solid tumors, VISTA expression is associated with poor overall survival and is also described as a potential mediator of resistance to anti-CLTA-4 and anti-PD1 therapies. VISTA expression was also investigated in hematological tumors and described as highly expressed on blasts including leukemic stem cells from AML patients. Therefore, VISTA is a unique and potentially new target for cancer immunotherapy in this indication. Kineta has developed a fully human monoclonal antibody targeting VISTA, KVA12123, currently evaluated in a Phase 1/2 clinical trial alone and in combination with pembrolizumab in cancer patients with advanced solid tumors. We also believe that VISTA could be a promising target for AML patients. Therefore, we have tested in vivo, in a Kasumi-3 AML disseminated tumor model, the efficacy of our antibody alone or in combination with standard of care therapy, cytarabine and doxorubicin. Like leukemic cells in AML patients, the Kasumi-3 cell line exhibits a high level of VISTA expression on its cell surface. Female NOD-scid mice were first inoculated with Kasumi cells via tail vein injection, and tumor engraftment was evaluated at different time points in the blood, spleen and bone marrow to monitor engraftment using CD34, CD33, CD45 and HLA-DR cell surface expression markers. Then, when tumor burden was high, standard of care treatment (5 doses of 16mg/kg AraC by IV injections and 3 doses of 0.5mg/kg Dox by IV injections) was initiated in indicated groups for 5 cycles, before starting treatment with KVA12 for the rest of the study. Tumor engraftment was tested after chemotherapy alone to evaluate the efficacy before starting treatment with our antibody. Our results showed that untreated mice had higher number of Kasumi-3 cells in the blood when compared to chemotherapy treated mice. Two backbone versions of our antibody were then tested, a human IgG1 - KVA12123 and a human IgG4 - KVA12402, at 30mg/kg IP injections twice a week for 5 weeks. Our data showed that KVA12123 and KVA12402 as single agents and in combination with chemotherapy reduced Kasumi-3 tumor load in the blood, spleen and bone marrow within the groups and significantly improved survival when compared to IgG1 control groups. These data all together indicate that anti-VISTA targeted therapy should be evaluated in myeloproliferative disorders like AML alone or in combination with standard of care.

CD123 As a Therapeutic Target in Accelerated and Blast Phase Myeloproliferative Neoplasms (MPNs)

Background: CD123 expression in MPNs has been reported in case series and CD123 has been a therapeutic target in one phase I study of patients with chronic myelomonocytic leukemia or myelofibrosis in chronic phase. CD123 has also been described as a marker of leukemic stem cells and incorporated in chimeric antigen receptor T-cell products for treatment of acute myeloid leukemia in clinical trials. Herein, we describe the clinical phenotype of patients with accelerated (AP) or blast phase (BP) MPNs paired with CD123 co-expression on CD45(dim) myeloid blasts. Methods: This is a retrospective, single-institution study of patients with AP and BP MPNs. Subjects were identified by search for MPN diagnoses in the Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank (OSUCCC-LTB). Relevant clinical data was collected by retrospective chart review with approval of Institutional Review Board. CD123 expression was measured by immunophenotyping of cryopreserved peripheral blood (PB) or marrow (BM) samples. Blasts were identified by CD45(dim) gating of lymphocytes. Lymphocytes were also gated on CD7, CD13, CD33, CD34, and CD117. CD123 expression was stratified by quartile and correlation with disease phase, symptoms, splenomegaly, transfusion-dependence, and molecular mutations measured by Chi-square test. Results: Forty-one PB or BM samples from 24 subjects were identified from the OSUCCC-LTB. Nine subjects had paired PB and BM specimens and 6 subjects had samples obtained at multiple timepoints in the course of their disease. Median age at time of sample collection was 65.8 years (range 35.2-79.7) and median time from initial diagnosis to sampling was 13.1 months (range 0-96.5). Constitutional symptoms were present at the time of collection in 24 specimens (58.5%) and splenomegaly present in 20 specimens (51.3%). At sample collection, 19.5% had never received treatment, 19.5% had been previously treated, and 61% were currently on treatment. Most patients on treatment (n=16, 64%) were receiving JAK inhibitor at sample collection. Nineteen samples were obtained in chronic phase (46.3%), nine in AP (22.0%), and thirteen in BP (31.7%). Median WBC count was 4.37K/uL (range 0.3-174.6), median peripheral blasts were 3.2% (range 0-76%), median hemoglobin was 8.8 g/dL (range 5.6-13.2), and median platelets were 42K/uL (range 5-564). Sixteen samples (39.0%) were from transfusion-dependent patients, of which, 68.8% were both red blood cell (RBC) and platelet transfusion-dependent, 25.0% were RBC transfusion-dependent only, and 6.2% were platelet transfusion-dependent only. Median lactate dehydrogenase at time of sampling was 300 U/L (range 116-2,142). Median bone marrow blasts at time of sampling were 3% (range 0-82%). Molecular sequencing and karyotype +/- 30 days of sample collection were available for 18 (43.9%) and 27 (65.9%) of specimens, respectively. The most common mutations identified were JAK2 (50%), ASXL1 (27.8%), SRSF2 (22.2%), CALR (16.7%), and TP53 (16.7%). Normal karyotype was reported in 37% of the samples (one was consistent with donor chimerism post-allogeneic SCT), 33.3% had complex karyotype, 11.1% had del 5q or 7q/-7, and 18.5% had del 13q or 20q. CD123 expression on the CD45(dim) gated blasts ranged from 0.3-95.6% with a median value of 11.02%. CD123 expression is presented in figure 1 box plot. Figure 2a shows correlation between CD123 expression and AP/BP compared to chronic phase disease (p < 0.01). Figure 2b shows CD123 expression correlation with ASXL1 compared to unmutated ASXL1 (p =0.023). There were no statistically significant associations between CD123 expression and any other tested variables. Conclusions: These single-institution data suggest that high CD123 expression on blasts is associated with leukemic transformation and high-risk disease. This is demonstrated by correlation of higher expression in AP and BP MPN as well as correlation with ASXL1 mutation. CD123 expression in MPNs has been previously reported and explored as a therapeutic target in a single phase I trial, though these data suggest that specifically targeting high risk disease, defined by blasts >10% or the presence of ASXL1 mutation, may be more effective with greater target availability. This is an especially important subpopulation of patients with MPNs in need of more effective and less toxic therapy than current standards of care that typically include cytotoxic chemotherapy.

Combination of Decitabine and Etoposide Is Highly Effective in Treating p53-Mutated MDS/AML Via Activating Notch Signaling

The elderly MDS/AML patients, especially those with TP53 mutations, remain very difficult to treat and resistant to chemotherapy. A total of 105 elderly (age above 60 years) high risk MDS (n = 84, IPSS-R score > 4.5) or AML-MRC (n = 21) patients' records were collected from three medical centers across China and retrospectively analyzed. Decitabine and low dose etoposide was found to be superior to standard decitabine alone in MDS/AML patients with higher ORR (70.0% vs 52.7%) after two cycles of treatment, the longer median durations of ORR (24.0 months vs 12 months) and CR (38.0 months vs 24.0 months), and longer event-free survival (13 months vs 9 months, p = 0.023), without significant improvement in overall survival (20 months vs 15 months, p = 0.059). Other than the TP53 mutation, no other gene mutation (including NRAS, DNMT3A, TET2, EZH2, ASXL1, RUNX1) in the D+E group could reliably predict OS and EFS when compared to wild type phenotype. Patients with TP53 mutations showed higher response rate after two cycles of D+E therapy and had prolonged overall survival (Mutation vs WT: 31 months vs 9 months, p=0.012) and event-free survival (Mutation vs WT: 24 months vs 5 months, p = 0.022) compared with p53 wild type patients in D+E group from multivariate survival analyses. P53 mutated patients receiving D+E treatment also had significantly improved OS (31 months vs 12 months, p = 0.00) and EFS (24 months vs 8 months, p = 0.00) compared to patients receiving decitabine treatment. Moreover, we found decitabine and etoposide greatly reduced TP53 mutant AML tumor burden in the NSG mice xenografts (tumor burden: 92% reduction in p53-null xenograft mice, 65% reduction in p53-wild-type xenograft mice on day 20 after leukemia cells administration, p < 0.0001). It was found that TP53 mutant cells and p53 gene-editing MOLM13 cells showed terminal neutrophil differentiation in the combo of decitabine and etoposide, demonstrating the expected changes in cell-surface CD11b expression and neutrophil functional assays such as phagocytosis. Combo of decitabine and etoposide showed much higher percentage of differentiation marker expression compared to single drug decitabine or etoposide treatment. However, neither the p53 gene-edited THP-1 cells nor the TP53 wild type cell lines displayed markers of terminal differentiation, such as elevated CD11b expression or phagocytosis. Furthermore, the percentage of CD11b-positive cells increased significantly in p53-mutated primary cells while insignificantly in TP53 wild type cells. Decitabine and etoposide induced significant differentiation thus killing p53 mutated myeloid tumor cells and primary patients' cells in vitro. We also uncovered the underlying mechanism that why D+E could induce significant differentiation in TP53 Mutant while not in TP53 wild type myeloid tumors. Notch1 signaling pathway was activated after D+E treatment in TP53 Mutant myeloid tumors, which was consistently correlated with drug-induced myeloid differentiation. Notch1 knockout or Notch inhibitor incubation abrogated drug induced differentiation in TP53 Mutant myeloid tumors. The activation of the Notch1 signaling pathway has been observed to induce disease regression and decrease leukemia stem cells in AML mice as well as significant apoptosis in the AML patient sample. We also found D+E could upregulate transcription factor transcripts such as ETS1, ETS2, which could bind specifically to PSEN1 (PS1) promoter and activate its transcription directly. PS1 represent catalytic subunit of γ-secretase complexes and is responsible for Notch recognition. While in TP53 wild type AML cells, p53 might interact with p300. p300 acts as a coactivator of Ets1 and Ets2, competition for binding to p300 with p53 may confront p300 recruitment and repress ETS transcription activation directly, which might exert its effect on PS1 repression, thus inhibiting γ-secretase complex activity. In short, integrated p53 could repress PSEN1 transcription thus interfering gamma secretase complex activity, Notch recognition and cleavage as well as abrogating drug induced Notch activation and myeloid differentiation, which could be achieved in p53 mutated state. Combination of decitabine and etoposide regimen is also relatively safe and tolerable in elderly patients, which may open up a new path of therapy in the p53 mutated AML/MDS.

Mesenchymal Stromal Cell-Derived MMP14 Facilitates Leukemia Progression and Chemotherapy Resistance

Abstract i ntroduction:Acute Myeloid Leukemia (AML) stands as a menacing hematological malignancy, characterized by the aberrant proliferation and development of myeloid cells, entailing formidable therapeutic complexities. Matrix metalloproteinases (MMP) represent pivotal enzymes that dismantle the extracellular matrix by disintegrating tissue barriers. Among these, MMP14 assumes significance as it governs the invasion and metastasis of cancer cells, exhibiting heightened expression in solid tumors, including prostate cancer, colon cancer, pancreatic cancer, breast cancer, and skin cancer, thereby endorsing the invasiveness and metastatic potential of angiogenic inflammatory cancer cells. Nonetheless, the effect of abnormal expression of MMP14 derived from mesenchymal stem cells (MSCs) in AML patients upon the bone marrow microenvironment and AML pathogenesis, along with its underlying mechanism, remains unexplored. Methods: AML-MSCs were extracted from bone marrow samples of AML patients through whole bone marrow adherent culture. Lentiviral vectors carrying cDNA of the MMP14 gene and empty lentiviral vectors were meticulously constructed and subsequently introduced into AML-MSCs. Brdu and CFU-F assayed the proliferative capacity of knockdown and control MSCs, and osteogenic induced differentiation was performed in vitro on both groups of MSCs.AML cell lines Kasumi-1 and Molm-13 were subjected to co-culture with AML-MSCs to evaluate the migratory and invasive capabilities of AML cell lines , while the proliferative activity of AML cell lines was assessed via BrdU. Following co-cultivation, AML cell lines were exposed to 1μM cytarabine for 48 hours, and cell viability was gauged using flow cytometry. Moreover, CD34+ cells were isolated from leukemia patients' bone marrow using magnetic beads and co-cultivated with the aforementioned MSC groups to explore the impact of MMP14 derived from MSCs on the resistance of leukemia stem cells to cytarabine. Results: Knockdown of MMP14 reduces MSC proliferation and colony-forming ability while also impeding MSC osteogenic differentiation. AML cell lines subjected to co-culture with the MSC knockdown group exhibited a remarkable reduction in both invasion and migration abilities. The BrdU assay demonstrated a significant decrease in the proliferation rate of AML cell lines within the MMP14 knockdown group. Moreover, following 48 hours of cytarabine administration, flow cytometric analysis revealed a lower proportion of viable cells in the knockdown group when compared to the control group. The same trend was observed in primary cells, as evidenced by a decreased percentage of proliferating CD34+ cells within the knockdown group, along with a reduced proportion of viable cells following cytarabine treatment. Conclusion: The MMP14 derived from MSCs fosters AML progression and confers chemoresistance. Our study provides valuable insights into the significance of MMP14 in AML drug resistance and underscores its potential as a promising preclinical target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

Results from a Phase (Ph) 1 Clinical Study of the All-Oral Regimen of CC-486 and Venetoclax for Relapsed and Refractory Acute Myeloid Leukemia

Background: Venetoclax (Ven) with azacitidine (Aza) is the standard of care for newly diagnosed AML patients who are unfit for intensive induction chemotherapy. It is well known that this treatment also has activity in the relapsed and refractory (R/R) setting. However, this regimen requires 7 consecutive daily subcutaneous or intravenous doses of Aza each month, which continues indefinitely. The relative inconvenience of this, and its negative impact on quality of life, may lead to its early discontinuation, which may decrease the efficacy or durability of this regimen. A more convenient all-oral regimen may decrease visits to an infusion center, improve quality of life, and potentially increase the efficacy of the regimen due to improved compliance. CC-486 is the oral formulation of Aza that is currently approved for post-induction chemotherapy maintenance in AML. Methods: This is a single center open label, Ph I study investigating CC-486 and Ven in R/R AML patients. In the dose escalation phase, subjects received CC-486 at one of two cohorts (200 mg PO days 1-14 and 300 mg PO days 1-14). Ven was given at the 400 mg/day PO regimen, for 28 days, after an initial intra-patient dose escalation per the standard of care. Using a 3+3 study design for these two cohorts, we aimed to determine the maximum tolerated dose (MTD) of CC-486 in combination with Ven. An expansion cohort is planned for 10 additional patients treated at the MTD. To assess whether CC-486 exerts similar effects in the leukemia stem cell population (LSCs) compared to conventional IV Aza, we retrospectively analyzed patient samples from cohort 1 with a patient sample treated with Ven/IV Aza outside of the study. We performed mass spectrometry-based metabolomics analyses in LSCs isolated from patient samples as well as single-cell RNA sequencing. Results: A total of 9 patients were accrued for this Ph I study (Figure 1A). The average age at the time of enrolment was 66, and median prior treatment regimens was 1 (range 1-3). Five patients had received Ven/Aza in prior treatments. There were no dose-limiting toxicity (DLT) events and the MTD was determined to be 300mg. The most common hematologic toxicities were Gr 3-4 neutropenia (n = 8, 89%), Gr 3-4 anemia (n = 7, 78%), and Gr 3-4 thrombocytopenia (n = 5, 56%). The most common non-hematologic toxicities were Gr 1-2 nausea (n = 5, 56%), fatigue (n = 2, 22%), and diarrhea (n = 2, 22%). Febrile neutropenia was also seen in two subjects (Gr 1-2 n = 1, 11%, Gr 3-4 n= 1, 11%). Best responses were complete remission (CR) (n = 2, 22%) partial remission (PR) (n = 1, 11%), and stable disease (SD) (n= 3, 33%). Three patients proceeded to an allogeneic stem cell transplant. Overall survival was 288 days, 95% CI: (39 days, 424 days). To determine whether CC-486/Ven exerts similar effects on the LSC population compared to conventional IV Aza/Ven, LSCs were isolated from the bone marrow of a responder and non-responder, and these were compared to LSCs from a patient who underwent treatment and responded to conventional IV Aza/Ven. Given subjects who achieved a CR in the study cleared their blasts by day 4 rendering no leukemia cells for analyses, samples used were from Subject 1 (stable disease with unchanged blasts), and Subject 2 (stable disease with significant blast reduction). Metabolomics assays performed in LSCs at day 4 and day 28 suggested mitochondrial dysfunction, decreased electron transport chain complex II activity and decreased glutathione levels in Subject 2 and conventional IV Aza/Ven but not on Subject 1. These findings are consistent with Pollyea et al. 2018 suggesting CC-486/Ven targets LSCs by perturbing complex II activity and OXPHOS in LSCs. Further, scRNA seq performed in Subject 1 and 2 showed gene expression patterns similar to those seen in patients who respond to Aza/Ven vs not, including a significant shift of BCL-2 to MCL-1 expression in the cell population harboring LSCs (Figure 1B). Conclusion: CC-486 and Ven is an all-oral regimen currently being investigated for the treatment of R/R AML. Our Ph I dose escalation demonstrated a favorable safety profile for this regimen with a MTD of 300mg CC-486. Responses were seen in both dose cohorts including 2 CRs and 1 PR, and patients have been successfully bridged to hematopoietic stem cell transplant. Analyses performed in patient samples from this study suggested a similar effect in LSCs in CC-486/Ven compared to conventional IV Aza/Ven.

The Role of over-Expressed β Globin in Driving Relapsed B - Cell Acute Lymphoblastic Leukemia (B-ALL)

Background: The prognosis for patients with relapsed acute lymphoblastic leukemia (ALL) remains suboptimal despite intensification of chemotherapy, immunotherapy and stem cell transplant. We and others have identified genetic and epigenetic alterations enriched at relapse that drive drug resistance and relapse. In our recent work mapping the epigenetic landscape of B-ALL, we discovered novel super-enhancers (SEs), or regions with increased H3K27 acetylation, enriched at relapse in a majority of patients, implicating a role in clonal evolution. One of these SEs was upstream of the β globin locus and lead to aberrant expression of hemoglobin beta subunit ( HBB) mRNA. HBB upregulation at relapse was also found in a large meta-analysis of gene expression data from pediatric diagnosis/relapse pairs. Of note, b globin is normally found in erythrocytes that interacts with other subunits to form functional hemoglobin. We hypothesized that HBB upregulation in blasts plays a role in relapse and aimed to determine whether it leads to drug tolerance, an early recovery phenotype and/or increased clonal potential. Methods: B-lineage REH, RS4;11 and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) HBB or an empty vector (EV) control. Real time PCR was performed to confirm RNA expression and Western Blot analysis was performed to detect protein expression. We generated additional cell lines with a mutation in the translation start codon and another leading to a premature stop codon to unequivocally interrupt protein expression. The generated cell lines were then plated and evaluated for differences in apoptosis via Annexin V assay and proliferation, in response to chemotherapeutic agents commonly used in the treatment of ALL. Clonogenic growth in vitro was also assessed using MethoCult TM media. To test the possibility that HBB acted in generating a persistor cell phenotype, cell lines were placed in mercaptopurine and prednisone for 10-14 days at two different concentrations and counted throughout drug exposure and for an additional 10-14 days following removal of drug. Results: We did not observe significant differences in chemosensitivity to various chemotherapies used in the treatment of B-ALL. However, we did observe less cells undergoing apoptosis in HBB expressing cells compared to control following prolonged treatment with 6-mercaptopurine, suggesting an increase in a small population of drug tolerant cells. However, this did not result in a difference in recovery time following removal of the drug. We also demonstrated that β globin overexpression led to increased clonal growth in methylcellulose compared to control (Fig. 1A, REH) and this observation was replicated in a second cell line (Fig. 1B, RS4;11). While we confirmed mRNA expression, we were not able to confirm protein expression by Western blot or by immunoprecipitation. We therefore hypothesized that b globin mRNA, rather than protein, mediates the observed increase in clonal growth. Interestingly, we observed the same phenotype of increased clonal growth (Fig. 1) in mutated cell lines, suggesting a catalytic role for β globin RNA in expanding a leukemia stem cell population. Conclusions: The increased clonal potential upon overexpression of HBB could be indicative of the ability to reconstitute a tumor from a small subpopulation following treatment. Additionally, our data suggests that protein is not required for increased clonal growth leading us to hypothesize that catalytic RNA is responsible. We are also currently analyzing RNAseq data from the cell lines and will compare differential gene expression between the empty vector and HBB expressing cells to discover downstream pathways impacted by HBB. We will be testing this hypothesis further by performing limiting dilution analysis in an immunocompromised mouse model (with David Teachey, Children's Hospital of Philadelphia).

Enrichment in Metabolic Pathway Activation Corresponds to Immunoglobulin Gene Diversity across B Cell Developmental Stages in B-Lymphoblastic Leukemia

Background: Intraclonal genetic variation in the immunoglobulin heavy chain variable gene (IGHV) has been widely documented in B-lymphoblastic leukemia (B-ALL), but its biologic significance as a presumed reflection of the B cell stage of leukemic transformation has not been systematically evaluated. Using ultradeep, targeted sequencing of the rearranged IgH gene in 22 patients with B-ALL, we previously demonstrated that there are subsets of patients with either extensive or minimal diversity in recombination activating gene (RAG)-mediated variable (V), diversity (D), and joining (J) gene rearrangements and that gene expression profiling (GEP) reveals enrichment of oxidative phosphorylation (OXPHOS), glycolysis, and fatty acid metabolism Hallmark pathways among cases with V-DJ rearrangement diversity (Fries et al. ASH 2022). We observed enrichment in these same metabolic signatures among leukemia cells with a pre-B cell phenotypic signature found to be predictive of relapse (Good et al. Nat Med 2018; Liu et al. ASH 2021). To understand the functional implications of this metabolic activation observed among cases with IgH diversity, we applied cytometry by time of flight (CyTOF) phenotyping to determine if IgH clonal composition reflects the metabolic and proliferative activation inherent to B cell stage of origin which may have relevance to disease behavior and outcome. Methods: Using viably-frozen, pre-treatment bone marrow (BM) and/or peripheral blood (PB) biospecimens from a cohort of 18 patients with B-ALL with known IgH clonal composition (7 with extensive V-DJ diversity, 8 without diversity, and 3 lacking any dominant clonal sequence), we performed deep phenotyping by mass cytometry and compared signaling pathway activation patterns between cohorts and compared to healthy controls. Samples were analyzed by mass cytometry with a 40-marker panel including B cell developmental phenotypic and metabolic proteins. Developmental classification was performed as previously described (Good et al. 2018). To test statistical significance in frequency of each subpopulation between two groups, we applied a multiple unpaired t-test with correction by Holm-Sidak method. Results: We discovered that all leukemia samples - regardless of IgH clonal composition - were significantly enriched for phenotypic features reflecting a pro-BII cell state, whereas enrichment of other more or less mature cell stages varied according to IgH composition. Developmental classification demonstrated that cases lacking a clonal IgH rearrangement (N=3) - presumed to have derived from a B cell stage preceding an initial DJ joining event - were enriched at the pro-BII population (P<0.0001), as were cases with extensive V-DJ diversity (N=7; P=0.019), and those lacking in diversity (N=8; P=0.0007) compared to healthy BM. However, cases with V-DJ diversity were uniformly enriched for OXPHOS, glycolytic, and pentose phosphate pathway (PPP)-associated protein expression compared to those without V-DJ diversity. In cells with a pro-BII phenotype, differentially expressed proteins between diverse and non-diverse cases included proteins essential for glycolysis (GLUT1, ENO1, PKM1), PPP (PGD, TKT), and OXPHOS (SDHA, CS, ATPA5, CytC). Conclusions: Cellular metabolism is a known hallmark of cancer proliferation, leukemia stem cell maintenance, and therapeutic escape, just as genetic diversification is integral to tumor evolution. Using primary B-ALL specimens, we show that metabolic pathway activation coincides with extensive RAG-mediated IGHV gene diversification across B cell developmental phenotypes. Contrary to the canonical assumption in B-ALL that V-DJ rearrangement and diversity is tied to developmental state, our analysis suggests that all cases are enriched at the proB-preB transition, but with differences in cellular metabolism that distinguish the diverse from homogeneous rearrangement state. These data suggest that IgH composition is not merely a passive genetic signature exploitable for tracking purposes in B-ALL, but may also inherently reflect underlying biology relevant to clinical outcomes.

Unraveling Clonal Evolution and Mechanisms of Treatment Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal FLT3 N676K

Our understanding of how individual mutations, whether present in all or just a subset of the leukemia cells, affect cellular response to therapy is limited. Herein, we investigate the impact of different therapies on survival, evolution, and resistance patterns in an acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3 and subclonal FLT3 N676K. Leukemic bone marrow expressing KMT2A::MLLT3-mCherry in all cells and FLT3 N676K-GFP in 40% of cells was re-transplanted into sublethally irradiated recipients. Upon engraftment, recipients were subjected to different treatments, including chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was administered for 28 days, while the control group received vehicle ( Fig 1a). Each treatment prolonged survival with a median latency of 30 days for chemotherapy 37.5 days for AC220, 42 days for chemotherapy+AC220, and 45 days for AC220+Trametenib, and 25.5 days for control mice. At sacrifice, all mice displayed splenomegaly and leukocytosis. The treatment impacted the evolution of the KMT2A:: MLLT3+ FLT3 N676K cells and they expanded to clonal dominance in control and chemotherapy-treated mice. However, three distinct patterns of evolution were detected in AC220-treated mice with the FLT3 N676K-containing cells either expanding to clonal dominance (>80% of KMT2A:: MLLT3+ FLT3 N676K), the FLT3 N676K-containing cells being eradicated (>80% of KMT2A::MLLT3-alone), or dual similar sized clones expressing KMT2A:: MLLT3-alone or KMT2A::MLLT3+ FLT3 N676K(Fig1 b).Eradication of the FLT3 N676K-cells was rare, predominantly observed upon AC220+Trametinib treatment and the frequency of dual clones increased when chemotherapy was followed by AC220, in line with treatment affecting the evolution of genetically distinct clones. RNA-sequencing was used to determine altered gene expression patterns upon resistance and unveiled three major clusters including 1) mice expressing KMT2A:: MLLT3-alone, 2) control and chemotherapy-treated mice, and 3) AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting potential resistance mechanisms through activation of the MEK/ERK-pathway. All leukemias had a myeloid Cd11b + phenotype. However, the control and chemotherapy-treated leukemias also displayed an aberrant expression of the lymphoid marker B220 and showed enrichment of gene sets correlating with stemness and oxidative phosphorylation, that suggested a switch in cellular phenotype and metabolic state. In contrast, AC220-treated leukemias lacked or had a low fraction of B220 + cells and showed enrichment of gene sets correlating with granulocyte-monocyte progenitors (GMP) and immune regulatory pathways (Fig 1 c). Acquired AC220-resistance mutations was rare (n=2), suggesting activation of alternative pathways upon resistance. Re-treatment of established cell lines from treated animals with AC220 showed that the cells were resistant. To uncover mechanisms of resistance at the cellular level, single cell RNA-sequencing was performed on four mice and revealed the emergence of distinct cellular subpopulations in AC220-treated mice, with a strong GMP signature and upregulation of the homeobox gene Six1(Fig 1d).Overexpression of Six1 in all AC220-treated leukemias was further confirmed by bulk RNA-sequencing. Interestingly, Six1 has previously been described as a regulator for leukemia stem cell maintenance in AML and under Wnt/ß-catenin signalling control. Subsequent drug assays showed AC220-treated cells exhibited sensitivity to Six1 pharmacological inhibition, suggesting a dependency on Six1, as well as sensitivity to Wnt/ß-catenin inhibition. Taken together, the treatment given affected survival and impacted evolution of the FLT3 N676K-leukemia cells. The general absence of acquired mutations in most mice suggests that target-independent mechanisms may underline acquired resistance in most mice, and we propose the Six1/Wnt/ß-catenin axis as a potential vulnerability upon AC220-resistance.

Pre-Existing Chromatin States Regulate KLF4 Binding to Eliminate Leukemia Stem Cells

Transcription factors collaborate with chromatin's epigenetic states to regulate cell fate decisions. While recent in vitro work using cryo-EM (Sinha et al., Nature, 2023) suggested that histone modifications could regulate the activity of pioneer transcription factors, it was not clear whether these findings applied in vivo, or if chromatin states could influence transcription factor binding to affect cell fate. Our previous work (Liu et al., Leukemia,2014; Wang et al., Nat Commun,2019) showed that reprogramming factors (Oct4, Sox2, Klf4, and cMyc, collectively known as OSKM) selectively eliminated leukemia cells in vivo, with minimal impact on normal Hematopoietic Stem and Progenitor Cells (HSPCs). This suggested that cell fates induced by OSKM could be influenced by pre-existing chromatin states. Building on this hypothesis, we profiled the pre-existing chromatin states of leukemia cells and HSPCs using MNase-ChIP-seq, ATAC-seq, and WGBS-seq. In our Tet-on-induced models, Klf4 activation resulted in the targeting of distinct gene sets and exhibited similar dynamic changes in chromatin accessibility, despite these regions sharing the same active chromatin state. Using machine learning algorithms, we identified H3K18ac –a feature of the active chromatin states preferred by Klf4– as a dominant factor influencing Klf4's genomic binding. We then altered H3K18ac through an enzymatically deficient SIRT7, which is an H3K18ac deacetylase, and examined where Klf4 binds. We observed an increase in Klf4 binding at sites where H3K18ac levels were elevated. This result suggested that Klf4 binding was influenced by changes in H3K18ac. We exposed human CD34+ cells, several leukemia cell lines, and samples from leukemia patients to ATPO-253. This small molecule, currently in phase 1 clinical trials, has been found to enhance the levels of the KLF4 protein. This treatment led to an increase in KLF4 protein levels and we observed activation of apoptosis genes in the tested leukemia cell lines that possessed high pre-existing H3K18ac levels. In addition, we observed a decline across all tested leukemia samples, while the population of CD34+ cells essentially remained stable. In conclusion, our work demonstrated that pre-existing chromatin states, such as H3K18ac, regulate Klf4's genomic binding, thereby influencing distinct cell fates in leukemia cells and HSPCs. This opens potential avenues for clinical applications in targeted leukemia cell clearance using KLF4 or ATPO-253.

Inflammation Shapes the Transcriptional Landscape of the Bone Marrow Microenvironment in Post-Transplantation Leukemia Relapses

Allogeneic Hematopoietic Cell Transplantation (allo-HCT) represents the most successful therapeutic option for many patients suffering from Acute Myeloid Leukemia (AML). Nevertheless, leukemic cells often find means to evade control from the donor-derived immune system and re-emerge. To date, three alternative and frequent mechanisms by which AML cells modify their features and escape immune control have been described: genetic loss of the mismatched HLA haplotype (HLA loss), downregulation of HLA class II molecules and upregulation of inhibitory ligands, all ultimately leading to abolished leukemia recognition by T lymphocytes. However, increasing evidence suggests that leukemic cells weave a much broader net of interactions, involving several other immune cell types within the bone marrow microenvironment. Still, how this complex crosstalk between malignant cells and the bone marrow niche may shape the features of leukemia post-transplant relapse remains unknown. By exploiting the high resolution provided by single-cell RNA-sequencing (scRNA-seq), we provide a detailed insight of the human bone marrow immune microenvironment and the changes that occur following allo-HCT, both when disease reemergence is prevented, and when it occurs. Using cryopreserved BM aspirates, we profiled a total of 145,420 mononuclear cells from 25 adult AML patients at the time of disease relapse; 56,831 cells from 5 post-transplant patients in complete remission (CR), each tested at two different timepoints (+90 and +365 days) and 45,279 cells from 6 healthy controls (HC). First, scRNA-seq allowed us to recapitulate known leukemia-intrinsic features of each relapse modality, such as HLA loss, down-regulation of HLA class II molecules and upregulation of inhibitory T cell ligands. Moreover, combining genotype and Copy Number Variation (CNV) inference algorithms, we were able to separate malignant cells from their healthy counterparts. Interestingly, HLA loss relapses exhibited a more immature profile, with a significantly higher leukemia stem cell signature (LSC17) (p-value < 0.0001, Wilcoxon rank-sum test) (Ng SW et al., Nature 2016), hinting that the hematopoietic cell of origin impacts on the mechanism of post-transplantation relapse. Leukemic cells also featured a high inflammation-associated gene score (iScore) (Lasry A et al., Nat Cancer 2023), as also evident in non-malignant HSPCs from relapsed patients, which showed an increased IFN-α signature compared to HC and CR. However, AML samples that couldn't be categorized by a known mechanism of relapse displayed a significantly lower iScore, suggesting that leukemia escape in these patients might not have had an immune-related driver (Figure 1a). We next sought to characterize specific BM immune compartments. We overall detected a lower frequency of CD4 T cells in relapsed patients as compared to HC and CR patients (p-value = 0.04, Wilcoxon rank-sum test). We annotated 5 major Natural Killer (NK) cell clusters (immature CD56 dim, mature CD56 dim, CD56 bright, proliferating NK, NKT cells) and a smaller subset of immature CD56 bright NK cells characterized by higher expression of IFN-related genes and NK exhaustion markers, which was defined as inflamed NK cluster. Inflamed NK cells were more represented in patients with upregulation of T cell inhibitory ligands compared to other relapsed patients (5.42% of total NK vs. 0.74%, respectively. p-value = 0.03, Wilcoxon rank-sum test), thus suggesting a shared mechanism of dysfunction signature between T and NK cell that may favor leukemia immune evasion (Figure 1b). In conclusion, we generated a comprehensive atlas of the BM immune microenvironment in AML post-transplantation relapses, showing that inflammation shapes immune subset composition and transcriptional profile in the BM, and that leukemia mechanisms of immune evasion are influenced by the surrounding milieu. Overall, while an in depth understanding of whether leukemia relapse is caused by an inflamed microenvironment, or vice versa, might only be achieved through longitudinal sampling from patients before and after transplant, our results here exemplify how the BM niche could be exploited to find novel vulnerabilities in AML relapses, to be specifically targeted by personalized therapeutic approaches.

Cladribine Combined with Homoharringtonine and Cytarabine Achieves a High Remission Rate in Adult Patients with De Novo Acute Myeloid Leukemia, Especially for Adverse-Risk Group: A Prospective, Single Center, Single-Arm, Phase 2 Study

Background: For adult de novo acute myeloid leukemia (AML), the standard 3+7 induction regimen has approximately 60% complete remission (CR) rate. Recently, BCL-2 inhibitor plus intensive chemotherapies as novel induction regimens have shown an above 90% composite complete remission (CRc) rate, however still have limited response effect in an adverse risk group, especially for patients with specific cytogenetic abnormalities such as KMT2A rearrangements, FLT3, RAS, and TP53 mutations. Multiple studies have shown that homoharringtonine (HHT) can target leukemia diseases that have switched their anti-apoptotic dependence from BCL-2 to MCL-1. We recently demonstrated that cladribine (CdA) could eradicate BCL-2 inhibitor-resistant monocytic leukemia stem cells driven by RAS mutants and KMT2A rearrangements. (Pei S, et al. Cancer Discovery. 2023) Together, these clinical and basic findings suggest a rationale for combining CdA with HHT to treat AML patients. In this study, we report the efficacy and safety of a newly designed “CHA” regimen composed of CdA, HHT, and cytarabine (AraC) in adult patients with de novo AML. Methods: This is a prospective, single-center, single-arm phase 2 trial. Newly diagnosed AML patients aged 18-59 years were enrolled. The diagnosis, risk classification, and response evaluation were determined according to ELN 2022 criteria. All enrolled patients received the CHA regimen: CdA (5mg/m2, days 1-3), HHT (2mg/m2, days 1-5), and AraC (100mg/ m2, days 1-7) administered intravenously. The response was evaluated on day 28 via bone marrow routine and minimal residual disease (MRD) measurement by flow cytometry. The primary endpoint was CRc (CR plus CRi). Secondary endpoints were MRD, overall survival (OS), disease-free survival (DFS), event-free survival (EFS), and adverse events. Bone marrow (BM) samples on day 1 and 7 and peripheral blood (PB) samples on days 1-7 were collected for laboratory flow analysis to monitor the kinetics of leukemia reduction. We also treated primary AML specimens with single, dual, and triple combinations of CdA, HHT, and Ara-C to evaluate their anti-leukemic activity in vitro. Statistical analyses were carried out using SPSS 24.0. Survival curves were prepared using the Kaplan-Meier method. A two-tailed P value <0.05 was considered statistically significant. This trial is registered on ClinicalTrials.gov (NCT05906914). Results: A total of 33 patients were enrolled from August 1, 2021, to April 15, 2023. The median age was 49 years. 33% (11/33) of patients were at favorable risk, 42% (14/33) were at intermediate risk, and 24% (8/24) were at adverse risk. After one course of CHA induction therapy, the CRc rate was 87% (27 of 31 patients), and the CR rate was 84% (26 of 31 patients). The CR rates in the favorable, intermediate, and adverse-risk groups were 82% (9 of 11 patients), 75% (9 of 12 patients), and 100% (8 of 8 patients), respectively. Notably, six out of six patients with KMT2A rearrangement and ten out of ten patients with RAS mutations achieved MRD-negative CR. The overall MRD negativity was 89% (24 of 27 patients with CRc). In 31 evaluable patients, four patients did not reach CR; three of them held FLT3 mutations. 30-days treatment-related mortality (TRM) was 6% (2 of 33 patients), and both died of sepsis. The median neutrophil recovery time (from the end of chemotherapy to the neutrophil counts ≥0.5×109/L) and platelets recovery time (from the end of chemotherapy to platelets counts ≥20×109/L) were both 11 days. With a median follow-up time of 13 months (IQR: 6-17 months), the one-year OS was 86%, 1-year DFS was 89%, and the 1-year EFS was 76%. Flow cytometry analysis of serially collected PB and BM samples from nine randomly selected patients revealed that an average of 91.0% and 94.7% of leukemia burden was cleared in the PB and BM by day 7, respectively, demonstrating a rapid and effective response induced by CHA. Finally, our in vitro assay demonstrated a synergistic effect of CdA, HHT, and Ara-C on primary AML specimens including three patients with adverse-risk. Conclusion: We propose the CHA regimen as a novel, effective, and safe chemotherapy regimen for newly diagnosed adult AML patients with remarkable potency in treating adverse-risk group patients, especially those with KMT2A rearrangements and RAS mutations that are otherwise resistant to BCL-2 inhibitor-based therapies, but a limited effect on FLT3mutated AML patients.

A Rapid Test to Evaluate the Tumor Differentiation State of Acute Myeloid Leukemias

Introduction: The combination of the hypomethylating agent Azacitidine with Venetoclax, a BCL2 inhibitor (Aza+Ven), is the frontline therapy for patients with acute myeloid leukemia (AML) who are elderly or unfit for intensive chemotherapy. However, identifying responsiveness to Aza+Ven a priori remains a clinical challenge.Aza+Ven effectively targets the primitive leukemia stem cell population (Pollyea, 2018), whereas resistance is observed in differentiated tumors (Pei, 2020). Further insights into the hierarchical organization of hematopoiesis have led to a definition of malignant AML cell types that follow the progression from hematopoietic stem cell to myeloid differentiated cells based on gene expression signatures, and these have been used to classify AML tumors (van Galen, 2019). Classification schemes using gene expression to capture variations in leukemic composition represent key biomarkers of response to targeted agents (Zeng, 2022; Bottomly, 2022). However, the prompt availability of nuanced cell state signatures to guide clinical decision making is limited by the laborious nature of performing and analyzing RNA-sequencing studies. Rapid AML stratification based on these classifications permits the possibility of identifying patients most likely to benefit from a drug or combination. Methods: To classify cell states based on transcriptomic signatures, we developed two assay formats, involving custom Nanostring and TaqMan Array card panels, for evaluation of tumor cell states for AML patients. Both formats contained gene sets representative of the six hematopoietic lineage states (van Galen, 2019) and both provide outputs that are compatible with the timeline for clinical decision-making. Assays were run according to the manufacturers' specifications, and enrichment of each of the six gene signatures was assessed using the Singscore stable method (Foroutan, 2018). Banked AML patient samples with matched RNA-seq and ex vivo Aza+Ven drug sensitivity data were used to validate the assay formats. Data obtained from the rapid assays were validated against cell states determined by deconvolution of bulk RNA sequence and compared with respect to clinical immunophenotype data where available. Results : Both the Nanostring and quantitative PCR-based assay formats detected gene expression representative of the appropriate cell state as previously determined by bulk RNA sequencing (Fig. 1A). Correlation of Aza+Ven ex vivo sensitivity with tumor cell state signatures revealed patterns where primitive but not differentiated cell states associated with sensitivity. In contrast, RNA isolated from healthy donor samples showed expression of all six cell state signatures. Across a cohort of 50 primary AML patient specimens, enrichment scores for each gene set correlated well with those derived for the same patients using deconvolution of bulk RNA-seq (Pearson r values: 0.785 to 0.944). Cell state characterization by rapid gene expression assays also aligned well with clinical immunophenotype, a routinely available diagnostic characterization of tumor blasts. Surface expression of primitive (CD117) and mature (CD11b) markers associated with Aza+Ven ex vivo sensitivity and resistance overall, respectively (p=0.03 & 0.04); however, ex vivo sensitivity could be significantly further stratified among samples expressing these markers when considering the gene expression-based enrichment scores for progenitor- and monocyte-like gene signatures (low vs high p-values: 0.02-0.05) (Fig. 1B). Analysis of matched samples from individual patients before and after treatment also showed cell state signature changes that reflected their temporal disease progression and correlated with differences in ex vivo drug sensitivity and clinical immunophenotype. Conclusion:Incorporation of cell state into prospective evaluations of AML patients provides an early indication of responsiveness to Aza+Ven. With rapid turnaround, assays such as these may augment treatment stratification and improve patient outcomes. These assays offer additional versatility to detect changes in AML cells after forced differentiation or acquired drug resistance.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.