Leukemic Progenitor Research Articles

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Dietary γ-mangostin triggers immunogenic cell death and activates cGAS signaling in acute myeloid leukemia

Immunogenic cell death (ICD), one of cell-death types through release of damage-associated molecular patterns from dying tumor cells, activates tumor-specific immune response and elicits anti-tumor immunity by traditional radiotherapy and chemotherapy. However, whether natural products could induce ICD in leukemia is not elucidated. Here, we report dietary γ-mangostin eradicates murine primary leukemic cells and prolongs the survival of leukemic mice. As well, it restrains primary leukemic cells and CD34+ leukemic progenitor cells from leukemia patients. Strikingly, γ-mangostin attenuates leukemic cells by inducing ICD as characterized by expression of HSP90B1, ANXA1 and IL1B. Additionally, γ-mangostin accelerates cytoplasmic chromatin fragments generation, promoting DNA damage response, and enhances cGAS activation, leading to up-regulation of chemokines. Meanwhile, it induces HDAC4 degradation and acetylated histone H3 accumulation, which promotes chemokines transcription. Ultimately, CD8+ T cell is activated and recruited by γ-mangostin-induced chemokines in the microenvironment. Our study identifies γ-mangostin triggers ICD and activates cGAS signaling through DNA damage response and epigenetic modification. Therefore, dietary γ-mangostin would act as a potential agent to provoke anti-tumor immunity in the prevention and treatment of leukemia.

Immune Surveillance of Acute Myeloid Leukemia Is Mediated by HLA-Presented Antigens on Leukemia Progenitor Cells.

The elimination of therapy-resistant leukemia stem and progenitor cells (LSC) remains a major challenge in the treatment of AML. This study identifies and functionally validates LSC-associated HLA class I and HLA class II-presented antigens, paving the way to the development of LSC-directed T cell-based immunotherapeutic approaches for patients with AML. See related commentary by Ritz, p. 430 . This article is featured in Selected Articles from This Issue, p. 419.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status

BackgroundAcute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)—the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.MethodsDigoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation—one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.ResultsPrimitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.ConclusionsOur data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery.53GxHmRcFt-KhVdMmSCJrsVideo

Ppm1d truncating mutations promote the development of genotoxic stress-induced AML.

Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.

Single-cell transcriptomics reveals multiple chemoresistant properties in leukemic stem and progenitor cells in pediatric AML

BackgroundCancer patients can achieve dramatic responses to chemotherapy yet retain resistant tumor cells, which ultimately results in relapse. Although xenograft model studies have identified several cellular and molecular features that are associated with chemoresistance in acute myeloid leukemia (AML), to what extent AML patients exhibit these properties remains largely unknown.ResultsWe apply single-cell RNA sequencing to paired pre- and post-chemotherapy whole bone marrow samples obtained from 13 pediatric AML patients who had achieved disease remission, and distinguish AML clusters from normal cells based on their unique transcriptomic profiles. Approximately 50% of leukemic stem and progenitor populations actively express leukemia stem cell (LSC) and oxidative phosphorylation (OXPHOS) signatures, respectively. These clusters have a higher chance of tolerating therapy and exhibit an enhanced metabolic program in response to treatment. Interestingly, the transmembrane receptor CD69 is highly expressed in chemoresistant hematopoietic stem cell (HSC)-like populations (named the CD69+ HSC-like subpopulation). Furthermore, overexpression of CD69 results in suppression of the mTOR signaling pathway and promotion of cell quiescence and adhesion in vitro. Finally, the presence of CD69+ HSC-like cells is associated with unfavorable genetic mutations, the persistence of residual tumor cells in chemotherapy, and poor outcomes in independent pediatric and adult public AML cohorts.ConclusionsOur analysis reveals leukemia stem cell and OXPHOS as two major chemoresistant features in human AML patients. CD69 may serve as a potential biomarker in defining a subpopulation of chemoresistant leukemia stem cells. These findings have important implications for targeting residual chemo-surviving AML cells.

Leukemic progenitor compartment serves as a prognostic measure of cancer stemness in patients with acute myeloid leukemia

We systematically investigate functional and molecular measures of stemness in patients with acute myeloid leukemia (AML) using a cohort of 121 individuals. We confirm that the presence of leukemic stem cells (LSCs) detected through invivo xenograft transplantation is associated with poor survival. However, the measurement of leukemic progenitor cells (LPCs) through invitro colony-forming assays provides an even stronger predictor of overall and event-free survival. LPCs not only capture patient-specific mutations but also retain serial re-plating ability, demonstrating their biological relevance. Notably, LPC content represents an independent prognostic factor in multivariate analyses including clinical guidelines of risk stratification. Our findings suggest that LPCs provide a robust functional measure of AML, enabling quantitative and rapid assessment of a wide range of patients. This highlights the potential of LPCs as a valuable prognostic factor in AML management.

BOS-371, a monoclonal antibody against IL1RAP: Characterization in preclinical models of AML.

7030 Background: IL1 receptor accessory protein (IL1RAP) is a cell surface protein overexpressed on leukemia stem cells (LSC) and progenitors in AML, CML and high-risk MDS. It is an indispensable co-receptor for the IL1 superfamily, but its role reaches beyond anti-apoptotic pathways. IL1RAP directly interacts with and potentiates signaling of FLT3 and c-KIT which have significant roles in AML pathogenesis. High IL1RAP is correlated with poor prognosis and IL1 blockade attenuates expansion of LSCs. Thus, targeting IL1RAP has emerged as a promising immunotherapeutic approach for myeloid malignancies. BOS-371 is a humanized monoclonal antibody targeting IL1RAP that has been Fc-engineered to enhance its capacity to induce antibody directed cell cytotoxicity (ADCC) and deplete IL1RAP-expressing AML cells. Methods: The preclinical activity of BOS-371 was characterized using multiple assays. In vitro analysis included: binding affinity in IL1RAP-expressing cell lines; inhibition of IL1-dependent signaling as measured by IL1a -stimulated secretion of IL6 from MRC5 lung cancer cells; measurement of ADCC in co-cultures of human NK cells with either IL1RAP-expressing cell lines or primary AML cells; and cell growth by monitoring metabolic activity of primary AML cells. In vivo efficacy of BOS-371 was determined in AML models: MV4-11 and ML2 cells (CDX) implanted in NOG and NOD-SCID mice, respectively, and patient-derived (PDX) leukemia cells implanted in NOG mice. Results: BOS-371 binds with high affinity to IL1RAP-expressing cells (average EC50 337pM) leading to potent inhibition of IL1 signaling (IC50 600pM). Co-culture assays in the presence of BOS-371 showed potent induction of ADCC with an EC50 of 60-70pM (cell lines) or 4-11pM (primary cells) and 80-100% cell lysis (both cell lines and primary cells). The ability of BOS-371 to impact AML cell growth exclusively through inhibition of IL1 signaling was measured in a panel of 15 primary AML samples. Incubation with BOS-371 led to IC50s ranging from 20-700nM in responsive samples. Evaluation of BOS-371 activity in vivo showed that administration of BOS-371 resulted in significant anti-leukemic activity. In CDX studies, BOS-371 treatment led to > 50% decrease in systemic disease and engraftment in bones and organs. The results observed in CDX AML models were reproduced in PDX mouse models of AML, where decreases in both peripheral ( > 70%) and bone marrow ( > 50%) disease burden in response to BOS-371 were observed. Conclusions: These findings demonstrate that BOS-371 binds with high affinity to IL1RAP, potently inhibits IL1 signaling and induces ADCC, leading to anti-leukemic activity in vitro and in vivo. The preclinical results support the clinical investigation of BOS-371 for the treatment of myeloid malignancies. A Phase 1 trial is currently in development.

Acute Myeloid Leukemia Stem Cells in Minimal/Measurable Residual Disease Detection.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by an abundance of incompletely matured or immature clonally derived hematopoietic precursors called leukemic blasts. Rare leukemia stem cells (LSCs) that can self-renew as well as give rise to leukemic progenitors comprising the bulk of leukemic blasts are considered the cellular reservoir of disease initiation and maintenance. LSCs are widely thought to be relatively resistant as well as adaptive to chemotherapy and can cause disease relapse. Therefore, it is imperative to understand the molecular bases of LSC forms and functions during different stages of disease progression, so we can more accurately identify these cells and design therapies to target them. Irrespective of the morphological, cytogenetic, and cellular heterogeneity of AML, the uniform, singularly important and independently significant prognosticator of disease response to therapy and patient outcome is measurable or minimal residual disease (MRD) detection, defined by residual disease detection below the morphology-based 5% blast threshold. The importance of LSC identification and frequency estimation during MRD detection, in order to make MRD more effective in predicting disease relapse and modifying therapeutic regimen is becoming increasingly apparent. This review focuses on summarizing functional and cellular composition-based LSC identification and linking those studies to current techniques of MRD detection to suggest LSC-inclusive MRD detection as well as outline outstanding questions that need to be addressed to improve the future of AML clinical management and treatment outcomes.

Abstract A39: Role of LARP1 in the leukemogenesis of Acute Myeloid Leukemia

Abstract Mammalian target of rapamycin (mTOR) is a strong driver of tumorigenesis in multiple types of tumors, however targeting mTOR for cancer therapy has yielded only limited success. This may be explained in part by signaling redundancy with other pathways, such as CDK9 mTOR-Like (CTORC) complexes, recently described by our group. One of the binders common to mTORC1 and CTORC2 is La related protein 1 (LARP1). LARP1 influences ribosome biogenesis by regulating translation of 5'terminal oligopyrimidine tract (5’ TOP) containing mRNAs, which often encode ribosomal proteins and translation factors. Here, we validate LARP1 as common target of mTORC1 and CTORC2 as well as investigate its influence on leukemogenesis. To explore LARP1’s role in leukemogenesis we utilized CRISPR/CAS9 technology to create OCI-AML5 and U937 LARP1 knockout (KO) cells. Loss of LARP1 expression significantly diminished proliferation potential of AML cell lines both in vitro as well as in vivo when implanted into the flank of athymic nude mice as xenografts. Additionally, primary CD34+ leukemia cells were transfected with LARP1 targeting or scrambled siRNA and leukemic progenitor growth was assessed in methocellulose colony formation assays. We observed suppression of leukemic progenitors colony forming ability in LARP1 siRNA transfected cells, compared to the Ctrl siRNA transfected cells. We also determined enhanced sensitivity of Larp1 KO cell lines to standard chemotherapy agents such Azacitidine, Cytarabine and Venetoclax. We employed polysome profiling to compare global translation levels between Larp1 KO and control U937 cells. Our polysomal profiling results show that KO of LARP1 in U937 cells reduces global translation and ribosome biogenesis as expected as we observed a suppression of both monosomal and polysomal peaks. In summary, Larp1 is important for leukemogenesis and influences their response to treatment with currently used chemotherapy agents. These observations together suggest that LARP1 is a promising target for development of novel antileukemia approaches. Citation Format: Dominik A Nahotko, Aneta H Baran, Mariafausta Fischietti, Elspeth M Beauchamp, Leonidas C Platanias. Role of LARP1 in the leukemogenesis of Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A39.

Glucose partitioning in the bone marrow micro-environment in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) cells metabolise glucose by glycolysis-based re-programming. However, how glucose uptake is partitioned between leukaemia cells and other cells of the bone marrow micro-environment is unstudied. We used a positron emission tomography (PET) tracer 18F fluorodeoxyglucose ([18F]-FDG) probe and transcriptomic analyses to detect glucose uptake by diverse cells in the bone marrow micro-environment in a MLL-AF9-induced mouse model. Leukaemia cells had the greatest glucose uptake with leukaemia stem and progenitor cells having the greatest glucose uptake. We also show the effects of anti-leukaemia drugs on leukaemia cell numbers and glucose uptake. Our data suggest targeting glucose uptake as a potential therapy strategy in AML if our observations are validated in humans with AML.

Abstract 1548: CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells

Abstract The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short, and overcoming therapeutic resistance has proven difficult. The presence of chemoresistant leukemic stem cells (LSCs), among other mechanisms, has been implicated as a driver in the observed relapse. Antibody-drug conjugates (ADC) are targeted cancer therapeutics that leverage antigen specificity to deliver a potent cytotoxic payload. Recently, ADCs have shown promise in the clinic, but can also present safety and efficacy concerns. Mylotarg, an FDA approved CD33-directed ADC used to treat Acute Myeloid Leukemia, was the first ADC to be approved. The therapeutic is active against most AML tumors but can lead to severe myelotoxicity given the expression of CD33 on normal myeloid progenitor cells. We leveraged a pharmaco-pheno-multiomic analysis of primary AML tumors to uncover a previously unexplored target expressed on both LSCs and AML blasts, but not normal hematopoetic progenitor cells or other normal tissue. The target also showed capacity to rapidly internalize and has activity of well-established oncogenic kinases associated with expression. A novel monoclonal antibody (IgG1) with favorable binding and biophysical characteristics was established against this novel target and conjugated to a highly potent cytotoxic agent, creating a novel ADC. This ADC shows strong binding across AML tumors with effective internalization and highly potent killing of AML tumors. Citation Format: Garima Kaushik, Bandana Vishwakarma, Marina Bell, Kakajan Komurov. CO-1008, an ADC program targeting leukemic progenitor and blast AML cells, but not normal hematopoetic progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1548.

Abstract 3860: ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism

Abstract Background: Primary or secondary resistance to venetoclax is pervasively associated with mutational/non-mutational activation of RAS/RAF/ERK pathway which confers metabolic alterations as a compensatory adaptation. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition followed by RNAseq analysis to identify molecular mechanisms responsible for overcoming resistance to venetoclax. Results: ERK1/2 inhibition using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029, was highly synergistic with venetoclax at inducing apoptosis in AML. NRAS-mutant OCI-AML3 and THP1 cells, that are inherently resistant to venetoclax, were sensitized to venetoclax when combined with ERKi with ~90% apoptosis compared to 20% by venetoclax alone (Combination Index = 0.008). Synergy was also confirmed using isogenic OCI-AML2 cells with lab-generated acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax (CI=0.6). Leukemia progenitor cells in primary AML samples were also depleted by the ven+ERKi combination (CI:0.03-0.23). In line with our in-vitro findings, treatment with ASTX029 as a single agent or in combination with venetoclax significantly inhibited leukemia burden and increased survival in an OCIAML3 xenograft model (p<0.05). Single agent ERKi and combination treatment using an NRAS-mutant PDX resulted in significantly impaired clonogenic potential and decreased expression of cMyc and CD44 in CD34+CD38- population as demonstrated by CyTOF, indicating depletion of progenitors. Transcriptomic profiling of OCIAML3 cells revealed upregulation of 6 pathways in response to venetoclax including oxidative phosphorylation (OXPHOS), electron transport chain, Myc targets, E2F targets, epithelial mesenchymal transition and KRAS signaling. Interestingly, these pathways were downregulated after ERKi+venetoclax treatment along with decrease in glycolysis and fatty acid metabolism (FDR<0.05). Since, venetoclax treatment significantly enriched for OXPHOS, we examined the metabolic effect of ERKi to overcome venetoclax resistance. Seahorse analysis showed increased basal and maximal respiration, and ATP production in OCIAML2 venetoclax resistant versus parental cells. Combining ERKi with venetoclax reduced OCR and ATP production in OCIAML3 and OCIAML2-venetoclax resistant cells. This was accompanied by an increase in membrane depolarization (p=0.001) as well as enhanced mitochondrial ROS (p<0.001). Conclusion: The inhibition of ERK1/2 alters mitochondrial metabolism and functional integrity to overcome resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Christopher Hindley, Kim-Hien Dao, Martin Sims, V. Lokesh Battula, Michael Andreeff, Gautam Borthakur. ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3860.

The interplay between telomeric complex members and BCR::ABL1 oncogenic tyrosine kinase in the maintenance of telomere length in chronic myeloid leukemia

PurposeChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by recurrent genetic aberration in leukemic stem cells, namely Philadelphia chromosome caused by reciprocal translocation t(9;22)(q34;q11). In our study, we analyzed the telomeric complex expression and function in the molecular pathogenesis of CML.MethodsWe employed CD34+ primary leukemic cells, comprising both leukemic stem and progenitor cell populations, isolated from peripheral blood or bone marrow of CML patients in chronic and blastic phase to analyze the telomere length and telomeric-associated proteins.ResultsThe reduction in telomere length during disease progression was correlated with increased expression of BCR::ABL1 transcript and the dynamic changes were neither associated with the enzymatic activity of telomerase nor with gene copy number and expression of telomerase subunits. Increased expression of BCR::ABL1 was positively correlated with expression of TRF2, RAP1, TPP1, DKC1, TNKS1, and TNKS2 genes.ConclusionsThe dynamics of telomere length changes in CD34+ CML cells is dependent on the expression level of BCR::ABL, which promotes the expression of certain shelterins including RAP1 and TRF2, as well as TNKS, and TNKS2, and results in telomere shortening regardless of telomerase activity. Our results may allow better understanding of the mechanisms responsible for the genomic instability of leukemic cells and CML progression.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

DNA Damage Response (DDR) Is Associated With Treatment-free Remission in Chronic Myeloid Leukemia Patients.

Preclinical evaluation of CD70-specific CAR T cells targeting acute myeloid leukemia.

Chimeric antigen receptor (CAR)-T cell therapy has achieved unprecedented success in treating hematopoietic malignancies. However, this cell therapy is hampered in treating acute myeloid leukemia (AML) due to lack of ideal cell surface targets that only express on AML blasts and leukemia stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs). We detectedtheCD70 expression on the surfaces ofAML cell lines, primary AML cells, HSC, and peripheral blood cells and generated a second-generation CD70-specific CAR-T cells using a construct containing a humanized 41D12-based scFv and a 41BB-CD3ζ intracellular signaling domain. Cytotoxicity, cytokine release, and proliferation in antigen stimulation, CD107a assay, and CFSE assays were used to demonstrate the potent anti-leukemia activity in vitro. A Molm-13 xenograft mouse model was established to evaluate the anti-leukemic activity of CD70 CAR-T in vivo. CFU assay was explored to assess the safety of CD70 CAR-T on HSC. CD70 heterogeneously expressed on AML primary cells, including leukemia blasts, leukemic progenitor, and stem cells, but not expressed on normal HSCs and majority of blood cells. Anti-CD70 CAR-T cells exhibited potent cytotoxicity, cytokines production, and proliferation when incubated with CD70+ AML cell lines. It also displayed robust anti-leukemia activity and prolonged survival in Molm-13 xenograft mouse model. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo. Our study reveals that anti-CD70 CAR-T cells are a new potential treatment for AML. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo, suggesting that future studies aiming to generate innovative combinatorial CAR constructs or to increase CD70 expression density on leukemia cell surface to prolong the life-span of CAR-T cells in the circulation will be needed in order to optimize CAR-T cell responses for AML.

Potential clinical use of azacitidine and MEK inhibitor combination therapy in PTPN11-mutated juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm of childhood. The molecular hallmark of JMML is hyperactivation of the Ras/MAPK pathway with the most common cause being mutations in the gene PTPN11, encoding the protein tyrosine phosphatase SHP2. Current strategies for treating JMML include using the hypomethylating agent, 5-azacitidine (5-Aza) or MEK inhibitors trametinib and PD0325901 (PD-901), but none of these are curative as monotherapy. Utilizing an Shp2E76K/+ murine model of JMML, we show that the combination of 5-Aza and PD-901 modulates several hematologic abnormalities often seen in JMML patients, in part by reducing the burden of leukemic hematopoietic stem and progenitor cells (HSC/Ps). The reduced JMML features in drug-treated mice were associated with a decrease in p-MEK and p-ERK levels in Shp2E76K/+ mice treated with the combination of 5-Aza and PD-901. RNA-sequencing analysis revealed a reduction in several RAS and MAPK signaling-related genes. Additionally, a decrease in the expression of genes associated with inflammation and myeloid leukemia was also observed in Shp2E76K/+ mice treated with the combination of the two drugs. Finally, we report two patients with JMML and PTPN11 mutations treated with 5-Aza, trametinib, and chemotherapy who experienced a clinical response because of the combination treatment.

Mechanisms of response and resistance to combined decitabine and ipilimumab for advanced myeloid disease

AbstractThe challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.

Near-Early T-Cell ALL with Overexpression of BAALC and BCL-2 Genes, Non-Responsive to High-Dose Chemotherapy and Hematopoietic Stem Cell Transplantation, but with Impressive Response to Venetoclax

Clinical and laboratory data are presented of a 29 year female patient with BAALC- and BCL-2-positive T-cell acute lymphoblastic leukemia developed from early leukemic progenitors which was treated unsuccessfully with high-dose chemotherapy and allogeneic stem cell transplant (allo-HSCT), but revealed an impressive effect of venetoclax. For reasons beyond our control, allo-HSCT with myeloablative fludarabine-busulfan (14 mg/ kg) conditioning regimen was performed at the peak of relapse when blast count in bone marrow aspirate was 77.5%, whereas level of BAALC-expressing earlier progenitors (EP) reached 186%. HSCT was ineffective, since the number of blasts on day+30 reached 86%, whereas the burden of BAALC-expressing EP decreased to the cutoff values. Later, at post-transplant stage, an impressive response to venetoclax coupled with hypomethylating agent was observed. Following allo-HSCT and combined venetoclax therapy a severe pancytopenia developed, which required the second transplantation of peripheral blood stem cells from her haplo-matched father. After transplantation hematopoietic recovery started on day 10+. Although bone marrow aspirate at this point was hypo cellular, it contained all types of granulocytic and erythroid elements and 2.2% of blasts only.

Inhibiting the biogenesis of myeloid-derived suppressor cells enhances immunotherapy efficacy against mammary tumor progression.

While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1-based (PD-1-based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8+ T cell responses. Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.