Leukemic Clone Research Articles

UCHMC 1812: A phase 1b trial of CPX-351 plus gemtuzumab ozogamicin for relapsed/refractory acute myeloid leukemia.

7024 Background: Gemtuzumab ozogamicin (GO) is effective in favorable-risk acute myelogenous leukemia (AML), while its activity is limited in higher-risk AML in part due to multi-drug resistance (MDR) mechanisms, particularly P-glycoprotein efflux pumps. CPX-351 provides a survival benefit in secondary AML, a disease characterized by high MDR activity. The objective of this trial is to utilize CPX-351 as a bulk cytoreduction agent, to first reduce the high MDR leukemia clones, followed by sequential treatment with GO in a more favorable leukemia environment. Methods: This was a multicenter phase Ib trial of CPX-351 plus GO in relapsed/refractory (R/R) AML patients with standard 3+3 dose escalation design (NCT03904251). Cohort A was treated with CPX-351 100 u/m2 on days 1, 3, and 5 plus GO 3 mg/m2 (max 4.5 mg) on day 7. Cohort B was treated with the same regimen plus an additional dose of GO 3 mg/m2 on day 4. The primary endpoint was the maximum tolerated dose (MTD), defined as < 2 dose limiting toxicities (DLTs) in the highest dose cohort. Secondary endpoints were IWG complete remission (CR) rate at day 28-42, liver veno-occlusive disease (VOD) rate diagnosed by the Baltimore criteria, and time of hematologic recovery (defined as ANC > 1000/uL and platelet count > 100,000/uL). Patients were enrolled at 4 University of California Hematologic Malignancies Consortium (UCHMC) centers: UC Los Angeles, UC San Francisco, UC Davis, and UC Irvine. Results: Thirteen participants were enrolled. The median age was 63 years (range 29-75). Eleven had relapsed disease, and two had refractory disease. Most received one prior line of therapy (n = 9). Seven had ELN 2022 adverse-risk disease at initial diagnosis, and two had intermediate-risk disease. The MTD was not reached. DLTs were observed in 1/6 participants in cohort A (grade 3 rash), and 1/6 participants in cohort B (grade 5 intracranial hemorrhage in the setting of severe thrombocytopenia). There were no cases of liver VOD. Of 12 evaluable participants, 4 achieved CR (33%), of whom 3 were negative for measurable residual disease by multiparametric flow cytometry, and 1 one achieved partial remission. The median age of responders was 30 years (range 28-65). The median time to return of normal hematopoiesis in those who achieved remission was 37 days (range 36-43). Three participants received allogeneic hematopoietic cell transplant (alloHCT) following investigational therapy: one remains in remission 34 months post-transplant, one died of infectious complications shortly after alloHCT, and one relapsed at day +60. Conclusions: CPX-351 plus two doses of GO 3 mg/m2 is a feasible treatment with acceptable toxicity and reasonable marrow recovery kinetics. However, only three participants went on to curative alloHCT. Further evaluation for efficacy in a larger patient population is needed to determine the utility of this regimen in R/R AML. Clinical trial information: NCT03904251 .

Venetoclax-based salvage therapy for adult patients with relapsed/refractory acute lymphoblastic leukemia.

Dysregulation of BCL-2 family members has been reported in acute lymphocytic leukemia (ALL), with various BH3-dependencies of the leukemic clone. We conducted a multicenter retrospective cohort of patients with relapsed/refractory B or T ALL, with ven-chemotherapy or ven-navitoclax combinations, to assess efficacy and safety. Seventeen patients were included in the analysis, median age was 32 years, with 6 B-ALL and 11 T-ALL patients. Nine patients received venetoclax combined with chemotherapy, and 13 patients received venetoclax in combination with navitoclax, vincristine and asparaginase, of which 5 were already exposed to venetoclax in previous lines. ORR was 55% and 46% among the ven-chemotherapy and the ven-navitoclax-chemotherapy, respectively. Most of the responders proceeded to an allogenic bone marrow transplant in both cohorts. The most common adverse effects of the ven-navitoclax combination were infectious complications and hepatotoxicity. Our data demonstrated the possible efficacy of ven-chemotherapy and ven-navitoclax in r/r ALL with moderate toxicity.

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [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 A05.

NGS-defined measurable residual disease (MRD) after initial chemotherapy as a prognostic biomarker for acute myeloid leukemia

Treated AML patients often have measurable residual disease (MRD) due to persisting low-level clones. This study assessed whether residual post-treatment somatic mutations, detected by NGS, were significantly prognostic for subsequent clinical outcomes. AML patients (n = 128) underwent both pre-and post-treatment testing with the same 42-gene MRD-validated NGS assay. After induction, 59 (46%) patients were mutation-negative (0.0024 VAF detection limit) and 69 (54%) had ≥1 persisting NGS-detectable mutation. Compared with NGS-negative patients, NGS-positive patients had shorter overall survival (17 months versus median not reached; P = 0.004; hazard ratio = 2.2 [95% CI: 1.3–3.7]) and a shorter time to relapse (14 months versus median not reached; P = 0.014; HR = 1.9 [95% CI: 1.1–3.1]). Among 95 patients with a complete morphologic remission (CR), 43 (45%) were MRD-positive by NGS and 52 (55%) were MRD-negative. These MRD-positive CR patients had a shorter overall survival (16.8 months versus median not reached; P = 0.013; HR = 2.1 [95% CI: 1.2–3.9]) than did the MRD-negative CR patients. Post-treatment persisting MRD positivity, defined by the same NGS-based test used at diagnosis, is thus a more sensitive biomarker for low-level leukemic clones compared to traditional non-molecular methods and is prognostic of subsequent relapse and death.

Abstract LB363: Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia

Abstract The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human cancers. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, “Complete Capture of Mutational Burden” (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines representing all major genetic groups of acute myeloid leukemia (AML). Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSGS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Citation Format: Andriana Kotini, Saul Carcamo, Nataly Cruz-Rodriguez, Malgorzata Olszewska, Dan Hasson, Eirini Papapetrou. Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB363.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract 392: Targeting drug-resistant acute myeloid leukemia (AML) clone using casein kinase II inhibitor

Abstract Introduction: Resistance to frontline cytotoxic therapy is rampant in AML. Molecular mechanisms promoting drug resistance are being identified to develop an effective strategy to overcome resistance. Protein kinase CK2 (casein kinase II) is a constitutively active, serine-threonine kinase that promotes cell survival and resistance to apoptosis in AML. A selective inhibitor of CK2, CX-4945 shows in vivo therapeutic efficacy in AML patient-derived xenografts. Here we report that CK2 kinase activity is high in AML cells resistant to cytarabine. Cytarabine-resistant cells are enriched for leukemia stem cells (CD34+/38-) and expansion of TP53 mutant clones. Specific inhibitors of CK2, CX4945 show selective cytotoxicity against the cytarabine-resistant cells. Methods: We developed cytarabine-resistant AML cell lines (MOLM-13, MV4-11, U937, Kasumi-1). We used TP53 mutant clone isolated and expanded from cytarabine resistant MV4-11 cell line. Gene expression of cytarabine-resistant AML cells and that of cell lines with and without CX-4945 treatment was analyzed using RNA sequencing. mRNA expression of genes was measured using qPCR and protein level was measured using western blot. Drug response and drug synergy were assessed using MTT assay. AML patient-derived xenograft was used to evaluate in vivo therapeutic efficacy of the combination of cytarabine and CX-4945. Results: Cytarabine-resistant cells have high expression of CK2. Gene expression analysis shows that the cytarabine metabolism gene is significantly altered in cytarabine-resistant cells and CX4945-treated AML cells. NT5C2 is a cytosolic nucleotidase that dephosphorylates monophosphates, preventing cytarabine from being phosphorylated to active form. hENT1 is a nucleoside transporter that brings cytarabine into the cells for metabolism. In cytarabine resistant cells, NT5C2 has been found to be high while hENT1 is low, preventing ideal cytarabine metabolism. CX4945 treatment decreased the expression of NT5C2 and increases expression of hENT1. Cytarabine sensitivity is restored following genetic inhibition (shRNA) or treatment with a CK2 inhibitor. AML xenograft mice treated with a combination of CX-4945 and cytarabine have decreased leukemia burden and prolonged survival compared to either drug alone. Conclusion: These results suggest that: 1) Overactive CK2 promotes AML drug resistance in leukemia stem cells; 2) CK2 inhibitor CX-4945 selectively targets cytarabine-resistant cells; 3) The potential role of CK2 in leukemia stem cell survival and drug resistance in vivo needs further evaluation. Citation Format: Katherine Mercer, Koby Duke, Rajesh Rajaiah, Muhammad Daniyal, Yi Qiu, Sinisa Dovat, Yasin Uzun, Lijun Zhang, Suming Huang, Morgann Klink, Chandrika Gowda. Targeting drug-resistant acute myeloid leukemia (AML) clone using casein kinase II inhibitor [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 392.

PYK2 is overexpressed in chronic lymphocytic leukaemia: Apotential new therapeutic target.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.

Measurable Residual Disease and Clonal Evolution in Acute Myeloid Leukemia from Diagnosis to Post-Transplant Follow-Up: The Role of Next-Generation Sequencing

It has now been ascertained that acute myeloid leukemias-as in most type of cancers-are mixtures of various subclones, evolving by acquiring additional somatic mutations over the course of the disease. The complexity of leukemia clone architecture and the phenotypic and/or genotypic drifts that can occur during treatment explain why more than 50% of patients-in hematological remission-could relapse. Moreover, the complexity and heterogeneity of clone architecture represent a hindrance for monitoring measurable residual disease, as not all minimal residual disease monitoring methods are able to detect genetic mutations arising during treatment. Unlike with chemotherapy, which imparts a relatively short duration of selective pressure on acute myeloid leukemia clonal architecture, the immunological effect related to allogeneic hematopoietic stem cell transplant is prolonged over time and must be overcome for relapse to occur. This means that not all molecular abnormalities detected after transplant always imply inevitable relapse. Therefore, transplant represents a critical setting where a measurable residual disease-based strategy, performed during post-transplant follow-up by highly sensitive methods such as next-generation sequencing, could optimize and improve treatment outcome. The purpose of our review is to provide an overview of the role of next-generation sequencing in monitoring both measurable residual disease and clonal evolution in acute myeloid leukemia patients during the entire course of the disease, with special focus on the transplant phase.

Patient specific real-time PCR in precision medicine - Validation of IG/TR based MRD assessment in lymphoid leukemia.

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.

Role of Notch2 pathway in mature B cell malignancies.

In recent decades, the Notch pathway has been characterized as a key regulatory signaling of cell-fate decisions evolutionarily conserved in many organisms and different tissues during lifespan. At the same time, many studies suggest a link between alterations of this signaling and tumor genesis or progression. In lymphopoiesis, the Notch pathway plays a fundamental role in the correct differentiation of T and B cells, but its deregulated activity leads to leukemic onset and evolution. Notch and its ligands Delta/Jagged exhibit a pivotal role in the crosstalk between leukemic cells and their environment. This review is focused in particular on Notch2 receptor activity. Members of Notch2 pathway have been reported to be mutated in Chronic Lymphocytic Leukemia (CLL), Splenic Marginal Zone Lymphoma (SMZL) and Nodal Marginal Zone Lymphoma (NMZL). CLL is a B cell malignancy in which leukemic clones establish supportive crosstalk with non-malignant cells of the tumor microenvironment to grow, survive, and resist even the new generation of drugs. SMZL and NMZL are indolent B cell neoplasms distinguished by a distinct pattern of dissemination. In SMZL leukemic cells affect mainly the spleen, bone marrow, and peripheral blood, while NMZL has a leading nodal distribution. Since Notch2 is involved in the commitment of leukemic cells to the marginal zone as a major regulator of B cell physiological differentiation, it is predominantly affected by the molecular lesions found in both SMZL and NMZL. In light of these findings, a better understanding of the Notch receptor family pathogenic role, in particular Notch2, is desirable because it is still incomplete, not only in the physiological development of B lymphocytes but also in leukemia progression and resistance. Several therapeutic strategies capable of interfering with Notch signaling, such as monoclonal antibodies, enzyme or complex inhibitors, are being analyzed. To avoid the unwanted multiple "on target" toxicity encountered during the systemic inhibition of Notch signaling, the study of an appropriate pharmaceutical formulation is a pressing need. This is why, to date, there are still no Notch-targeted therapies approved. An accurate analysis of the Notch pathway could be useful to drive the discovery of new therapeutic targets and the development of more effective therapies.

Mathematical modeling of leukemia chemotherapy in bone marrow

Acute Lymphoblastic Leukemia (ALL) accounts for the 80% of leukemias when coming down to pediatric ages. Survival of these patients has increased by a considerable amount in recent years. However, around 15 20% of treatments are unsuccessful. For this reason, it is definitely required to come up with new strategies to study and select which patients are at higher risk of relapse. Thus the importance to monitor the amount of leukemic cells to predict relapses in the first treatment phase. In this work, we develop a mathematical model describing the behavior of ALL, examining the evolution of a leukemic clone when treatment is applied. In the study of this model it can be observed how the risk of relapse is connected with the response in the first treatment phase. This model is able to simulate cell dynamics without treatment, representing a virtual patient bone marrow behavior. Furthermore, several parameters are related to treatment dynamics, therefore proposing a basis for future works regarding childhood ALL survival improvement.

HTLV-1 persistent infection and ATLL oncogenesis.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus; whereas HTLV-1 mainly persists in the infected host cell as a provirus, it also causes a malignancy called adult T-cell leukemia/lymphoma (ATLL) in about 5% of infection. HTLV-1 replication is in most cases silent in vivo and viral de novo infection rarely occurs; HTLV-1 rather relies on clonal proliferation of infected T cells for viral propagation as it multiplies the number of the provirus copies. It is mechanistically elusive how leukemic clones emerge during the course of HTLV-1 infection in vivo and eventually cause the onset of ATLL. This review summarizes our current understanding of HTLV-1 persistence and oncogenesis, with the incorporation of recent cutting-edge discoveries obtained by high-throughput sequencing.

Nanopore sequencing of clonal IGH rearrangements in cell-free DNA as a biomarker for acute lymphoblastic leukemia.

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer, and patients with relapsed ALL have a poor prognosis. Detection of ALL blasts remaining at the end of treatment, or minimal residual disease (MRD), and spread of ALL into the central nervous system (CNS) have prognostic importance in ALL. Current methods to detect MRD and CNS disease in ALL rely on the presence of ALL blasts in patient samples. Cell-free DNA, or small fragments of DNA released by cancer cells into patient biofluids, has emerged as a robust and sensitive biomarker to assess cancer burden, although cfDNA analysis has not previously been applied to ALL. We present a simple and rapid workflow based on NanoporeMinION sequencing of PCR amplified B cell-specific rearrangement of the (IGH) locus in cfDNA from B-ALL patient samples. A cohort of 5 pediatric B-ALL patient samples was chosen for the study based on the MRD and CNS disease status. Quantitation of IGH-variable sequences in cfDNA allowed us to detect clonal heterogeneity and track the response of individual B-ALL clones throughout treatment. cfDNA was detected in patient biofluids with clinical diagnoses of MRD and CNS disease, and leukemic clones could be detected even when diagnostic cell-count thresholds for MRD were not met. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient, even when blasts are not physically present in the biofluid sample. The Nanopore IGH detection workflow to monitor cell-free DNA is a simple, rapid, and inexpensive assay that may ultimately serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

AML-derived extracellular vesicles negatively regulate stem cell pool size: A step toward bone marrow failure

Purpose of the studyLong-term repopulating hematopoietic stem cells (LTR-HSCs) have been previously shown to reside in close proximity to osteoblasts, where they take shelter in the bone marrow (BM) microenvironment against cytotoxic and apoptotic stimuli. Nevertheless, the function of the HSC niche is believed to undergo an adaptive evolutionary modification during leukemogenesis. Recent studies have demonstrated that leukemic clones can impact BM homing through extracellular vesicle (EV) secretion. However, the exact mechanism driving BM conversion is still unclear. In the present study, the human osteoblast cell line (MG-63) were subjected to various concentration of sera-derived EVs of patients with acute myeloid leukemia (AML) and healthy volunteers to assess if they are associated strongly enough to alter the expression pattern of cross-talk molecules involved in niche interactions. MethodTo gain a brief insight into the EVs secretion criteria, we first conducted a comparative analysis of sera-derived EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM), and Bradford assay. After incubating MG-63 cell lines with increasing concentrations of the EVs, Trypan-blue and microculture tetrazolium test (MTT) assays were used to evaluate the cell survival, logarithmic growth, and metabolic activity. Finally, the expression levels of OPN, ANGPT-1, and JAG-1 transcripts were evaluated through the qRT-PCR technique. ResultsHere, we report that AML-derived EVs can affect the viability, cell growth, and metabolic activity of the human osteoblasts cell line (MG-63) compared to those that received healthy-derived EVs. We also found that leukemic EVs tend to induce overexpression of OPN but reduce the expression of ANGPT-1 and JAG-1 genes in the osteoblast transcriptome, which may provide a potential context imposing selective suppression of HSC pool size. ConclusionThese findings extend the general concept of a novel mechanism in which leukemic EVs would make it possible to create a specialized pre-metastatic microenvironment in the interest of tumor expansion, allowing leukemic clones to overcome their HSCs counterparts.

Single Cell Genotypic and Phenotypic Analysis of Measurable Residual Disease in Acute Myeloid Leukemia

Measurable residual disease (MRD) represents the critical population of leukemic cells which persists following anti-leukemic therapy. The presence of MRD has powerful prognostic relevance and represents the reservoir responsible for acute myeloid leukemia (AML) relapse. It is therefore critical to enumerate and molecularly characterize MRD clones in order to better risk stratify AML patients and guide further therapy aimed at preventing relapse and improving outcomes. Current MRD methods, including multi-color flow cytometry (MFC) analysis and bulk NGS sequencing, face challenges in both sensitivity and specificity and are insufficient to distinguish between pre-leukemic and leukemic clones. Consequently, the imperfect predictive value of these techniques hinders meaningful clinical intervention. To overcome these shortcomings associated with conventional methods of MRD detection, we developed a novel single-cell (sc) MRD assay with high sensitivity and specificity by combining precursor/blast population enrichment with integrated scDNA sequencing and immunophenotypic analysis. We reasoned that the MRD clones responsible for relapse largely reside in immature compartments. Enriched CD34+ and/or CD117+ progenitors from 5 unique patient samples were multiplexed followed by integrated single cell analysis on the MissionBio TapestriV2 scDNA+protein platform. We developed a novel computational approach to deconvolute multiplexed sequencing reads and accurately assign cells to individual patients via K-means clustering and UMAP dimension reduction of germline SNP allele frequencies. To demonstrate the sensitivity and specificity of our scMRD assay, 10,000, 1,000, 500, and 200 AML blasts from 3 patients harboring known pathologic mutations (NRAS/RUNX1, JAK2/IDH2, and IDH2 alone) were individually spiked into 10 million normal bone marrow cells to test a sensitivity threshold of 0.1%, 0.01%, 0.005%, and 0.002%. Samples were FACS-enriched for blasts, multiplexed, subjected to the TapestriV2 microfluidics platform, and sequenced. The expected pathologic mutations were reliably identified in patients at a threshold of 0.01%, but not in normal subjects, demonstrating that our assay is nearly 10-fold more sensitive than the current ELN MRD standard [Fig 1]. We then applied our assay to 30 banked post-induction MRD samples from 29 patients (median age 71 years old, 15 male/14 female) who had a diagnosis of AML [Fig 2]. Overall, genomic analysis was concordant in 22/30 (73%) samples between bulk NGS (at MRD stage) and scMRD. The mean VAF of mutations was higher by scMRD than bulk NGS, consistent with our hypothesis that mutations are enriched in immature compartments. 17 mutations were identified by scMRD but missed/unreported by bulk NGS, including RUNX1 (n=5), NPM1 (n=3), KRAS (n=2), IDH2 (n=2), WT-1 (n=2), JAK2, TP53 and SRSF2 mutations, 14 of which were present at relapse. Clonal architecture was illustrated by scMRD data with identification of pre-leukemic and leukemic clones. In 5 post-HSCT samples, scMRD readily distinguished between donor vs. host cells and showed higher host cell chimerism compared to conventional STR testing, suggesting that host cells have higher enrichment in immature cell types and represent an early indicator of relapse. Our scMRD assay also enabled single cell immunophenotypic analysis, and revealed concordant abnormal immunophenotype of leukemic blasts identified by MFC. More importantly, differential immunophenotypic states were identified when comparing pre-leukemic and leukemic clones (i.e. DNMT3A only vs co-mutant DNMT3/NPM1) within and between patients. These data show that integrated genomic/immunophenotypic analysis at the MRD timepoint can distinguish between clonal hematopoiesis and pre-leukemic/leukemic clones with a substantively higher likelihood of relapse. In conclusion, our scMRD assay not only enhances MRD detection, but also provides a molecular glimpse into the clonal architecture of pre-leukemic/leukemic cells surviving AML therapy. Larger studies are needed to comprehensively elucidate the clonal architecture of MRD and study the dynamics of clonal evolution by comparing clonal architecture and immunophenotype at diagnosis and relapse. Our study paves the road for identifying/eradicating high-risk MRD clones and preventing relapse through a better understanding of the molecular drivers of MRD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunophenotypic Switch in Leukemic Clone Is Very Common at the Time of Post-Transplant Relapse of Acute Myeloid Leukemia While Immunophenotypic Profile at Diagnosis Correlates with Adverse Outcomes after Post-Transplant Relapse

Immunophenotypic Switch in Leukemic Clone Is Very Common at the Time of Post-Transplant Relapse of Acute Myeloid Leukemia While Immunophenotypic Profile at Diagnosis Correlates with Adverse Outcomes after Post-Transplant Relapse

Orthogonal Proteogenomic Approaches Identify the Druggable PA2G4-MYC Axis in 3q26 AML

Despite next-generation technologies improved our understanding of the complexity of high-risk acute myeloid leukemia (AML), effective targeted therapies are delayed due to the prevalence of major oncogenic disease drivers such as RAS, RUNX1, TP53. In some cases, the integration of orthogonal omics led to the identification or repurposing of drugs for "undruggable" transcription factors (TFs), prospecting a benefit for patients with the most aggressive leukemia subtypes. Here, we sought to discover how to target the Ecotropic Viral Integration Site-1 gene (EVI1/MECOM), the most lethal oncogenic TF in AML carrying chromosome 3q26 abnormalities. To this end, we intersected phenotypic, gene expression-based and proteomic high-throughput approaches for small molecule and target identification. We screened a collection of 5294 compounds enriched for Food and Drug Administration (FDA) approved or bioactive drugs at 1 µM and identified those that caused more than 95% reduction of cell viability compared to vehicle control in MOLM1 and UCSD/AML1 cell lines carrying 3q26 rearrangments (EVI1high). We then selected a set of sentinel genes that defined an EVI1 "on" and "off" transcriptional signature based on the TF1 EVI1high cell line treated with an EVI1-directed siRNA or control, and used the Connectivity Map to identify inducers of an EVI1 "off" state. Histone deacetylase inhibitors (HDACi) scored as the top class of compounds able to repress leukemia proliferation by suppressing the EVI1 signature. We focused on the compounds with pan (AR-42, belinostat) or selective (entinostat) HDAC inhibitory properties. These molecules induced apoptosis and decreased the levels of EVI1 in AML cell lines and in primary EVI1high blast cells. Interestingly, the expression of EVI1 sensitizes cells to HDAC inhibition both in an inducible EVI1 cell model and in EVI1high compared to EVI1low AML cell lines. Primary EVI1high blasts were also more sensitive to HDAC inhibition compared to those from other AML subtypes, while equally resistant to chemotherapy agents. We translated these results in a "N-of-1" clinical trial proposing entinostat in association with azacytidine to patients with relapse/refractory 3q26 AML based on a compassionate use program for this regimen. Pharmacodynamic modulation of EVI1 was assessed at 6 hours (T6) after entinostat administration in patients and in matched patient-derived AML xenografts (PDLX). EVI1 expression markedly decreased in circulating blasts collected at T6 compared to T0 (before treatment), and EVI1 depletion paralleled clinical and hematologic improvements at follow-up evaluations. This effect was not observed in patients treated with cytarabine. Longitudinal single-cell RNA sequencing (scRNASeq) of bone marrow AML cells collected before and after patients' treatment cycle revealed an inhibitory effect on Myc target genes in the leukemic clone. This effect was also observed in cell lines treated with AR-42, belinostat and entinostat suggesting that different HDACi regulates the Evi1-Myc axis in 3q26 AML. Not surprisingly, HDACi mediated or genetic silencing of EVI1 led to a decrement of MYC in EVI1high AML models. To dissect the EVI1 co-transcriptional complex and explain for the modulation of Myc signaling in EVI1high AML, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME). Sixty-nine proteins co-immunoprecipitated with endogenous EVI1 both in cell lines and in 3q26 PDLX models. Eleven of these EVI1 interactors were targets of Myc signaling, including PA2G4, a RNA-binding protein that was markedly modulated by HDACi. Importantly, PA2G4 silencing or inhibition (WS6) depletes MYC at transcriptional and protein levels recapitulating the phenotypic effect seen with HDACi in EVI1high cell models. Consequently, WS6 treatment reduced the growth of AML cells in PDLX models in vivo by suppressing an EV1-MYC signature at single-cell leukemia level without inducing widespread toxicities. In contrast, PA2G4 overexpression rescued EVIhigh AML cell lines from the effects induced by HDACi inhibition. These data support the evidence that PA2G4 is a mediator of Evi1-Myc axis in AML. In conclusion, our work positions PA2G4 at the crosstalk of the Evi1-Myc leukemogenic signal for developing new therapeutics and, at this interesting time, urges upfront HDACi-based combination therapies in patients with 3q26 AML.

Unraveling at Diagnosis Chemoresistant Relapse-Initiating Clones in Acute Myeloid Leukemia: Combined Functional and Genomic Analyses Using Single-Cell-Derived Hematopoietic Stem and Progenitor Cell Subpopulations

Unraveling at Diagnosis Chemoresistant Relapse-Initiating Clones in Acute Myeloid Leukemia: Combined Functional and Genomic Analyses Using Single-Cell-Derived Hematopoietic Stem and Progenitor Cell Subpopulations

First Description of a Frameshift PAX5 Germline Variant in Two Siblings with B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: Recent studies have highlighted that about 5% of children with Acute Lymphoblastic Leukemia (ALL) carry a germline mutation that predispose to leukemogenesis. PAX5 somatic mutations are frequently identified on sporadic B-ALL, but recently pathogenic PAX5 germline variants have been described in families with recurrence of B-ALL (Shah et al. 2013; Auer et al. 2014; Yazdanparast et al. 2015, Duployez et al. 2021). We describe the case of two siblings both affected by B-ALL with evidence of a new familial PAX5 variant. Case A was a male, 13 years old, diagnosed with B-II ALL. He was enrolled into the AIEOP-BFM ALL 2009 treatment protocol and according to minimal residual disease (MRD) levels he was stratified as medium risk. Eight years later, his sister (14yrs, named as case B) was diagnosed with B-II ALL, and stratified in the medium risk group of AIEOP-BFM ALL 2017 protocol. The two patients were otherwise healthy and borne from healthy unrelated parents; no history of hematological malignancies was reported across the pedigree. Methods: We performed a custom Next Generation Sequencing ALL predisposition panel on DNA extracted from bone marrow samples of both disease onset and remission phase (defined as negativity by PCR-MRD, 10-4 sensitivity). The libraries were performed following Nextera Flex for Enrichment and sequenced on Illumina Nextseq550 platform; the analyses were carried out by Congenica software. The variants of interest were validated and investigated in the family pedigree through PCR and Sanger sequencing. Digital-MLPA testing 55 ALL hot spot genes (including all PAX5 exons) was performed. Results: Through the NGS analysis, we showed that both patients shared a novel germline heterozygous PAX5 variant (c.548delG, p.Gly183AlafsTer84); moreover, the disease samples of both patients carried an additional somatic PAX5 variant (c.239C>G, p.Pro80Arg; variant fraction: 29% in Case A and 38% in Case B). The Digital-MLPA using the ALL panel, excluded the presence of shared copy number alterations (CNV) and, specifically, did not identified CNV alterations involving the PAX5 locus. The familiar segregation showed a paternal origin of the germline PAX5 variant (p.Gly183AlafsTer84); the mother was found to be wild-type. The PAX5 variant was also found in the DNA sample of the patients' grandmother of the paternal branch; a healthy brother of probands (24 years old) was not tested. To investigate a possible impairment of PAX5 function in the development of the B-cell lineage, we evaluated in both parents the B-cell repertoire on peripheral blood fresh mononuclear cells by FACS analysis. Interestingly, the father has a significant reduction on mature IgD+/CD27+ B-cells with a concomitant accumulation of IgD+/CD27- naïve B-cells. Conclusions: We are reporting a novel germline PAX5 p.Gly183AlafsTer84 in a family with recurrence of B-ALL. In contrast to the previous PAX5 described cases that carry an inherited missense variant resulting in a modest attenuation of PAX5 activity, our patients present a germline frameshift variant that brings to premature stop of the protein, suggesting a more destructive impact on PAX5 activity. As in the other reported cases, our patients presented an additional somatic event on PAX5 that further compromise PAX5 function, leading to the establishment of the leukemic clone. Interestingly and different from the literature, the two siblings share the same PAX5 p.Pro80Arg somatic variant, that has been characterized by Gu as a pathogenic variant impairing B lymphoid development and promoting leukemogenesis (Gu et al. 2019). The detailed molecular characterization, including Whole Transcriptome NGS analysis, is ongoing to investigate whether a PAX5-specific signature can be identified. Moreover, a single-cell NGS approach on remission samples of the two siblings could help to identify specific important pathways and altered gene suppression profiles, preceding full leukemia. Consent-dependent characterization of additional family members are ongoing to assess the penetrance of leukemia associated to this new variant and possible additional features. The identification and characterization of germline PAX5 variants is of high interest in the context of leukemia predisposition because of its clinical implications in terms of familial genetic counselling and because of its contribution in the understanding ALL pathogenesis.