Therapy For Myeloid Leukemia Research Articles

What’s New in the Classification, Diagnosis and Therapy of Myeloid Leukemias

Myeloid leukemias are a broad group of hematological disorders, characterized by heterogeneous clinical and biological features. In recent years, unprecedented genetic discoveries and clinical–biological correlations have revolutionized the field of myeloid leukemias. The most relevant changes have specifically occurred in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia (CML) and myeloid neoplasms (MNs) with eosinophilia. The recently published International Consensus Classification (ICC) of myeloid neoplasms has addressed these changes, providing an updated framework and revised diagnostic criteria for such entities. This is also the aim of the 5th edition of the WHO classification of hematopoietic tumors, whose preliminary version was published in 2022. Parallel to this, new therapeutic options and novel molecular targets have changed the management of many myeloid entities, including AML and CML. This review aims to address the most relevant updates in the classification and diagnosis of AML, CMML, CML and MNs with eosinophilia. The state of the art of treatment and future therapeutic options for such disorders are also discussed.

Abstract 5662: Targeting N-myristoylation for therapy of adult acute myeloid leukemia

Abstract Two N-myristoyltransferases (NMTs) NMT1 and NMT2 catalyze the reaction. NMT1 is ubiquitous expressed and is essential for cell survival while NMT2 is more variably expressed and non-essential suggesting that their substrate specificity and activity levels differ. Historically, inhibition of myristoylation was suggested as a therapeutic anti-cancer target since NMTs expression were shown to be increased in numerous types of cancers and myristoylation was shown to be essential for proper localization and activity of some important proto-oncogenes such as Src Family Kinases (SFKs). Recently, we showed NMT2 expression is lost in numerous haematological cancer cell lines (including AML) and that these haematological cancer cell lines are exquisitely sensitive to the pan-NMT inhibitor PCLX-001. PCLX-001 recently entered human clinical trials as once daily oral therapy for relapsed/refractory B-cell Non-Hodgkin Lymphoma and advanced solid malignances. Dysregulation and oncogenic activity of SFKs occurs frequently in AML, suggesting NMT inhibition could provide therapeutic benefit in this indication. Data analysis from the TCGA transcriptome database revealed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML. Moreover, high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, reduced levels of SFK proteins and SFK phosphorylation as well as significant increases in ER stress marker BIP protein and caspase 3 cleavage. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations that spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. PCLX-001 monotherapy had dose-dependent anticancer activity in an AML MV-4-11 cell line derived xenograft (CDX) and two AML patient derived xenografts (PDXs) and produced complete remissions in subcutaneous AML CDX. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. PCLX-001 preferentially targeted AML cells inducing apoptosis and reducing leukemic burden. These findings validate NMT inhibition as a novel therapeutic strategy for AML and warrant the evaluation of PCLX-001 in clinical trials for adult AML. Citation Format: Jay Gamma, Aishwarya Iyer, Megan Yap, Zoulika Zak, Krista Vincent, Cassidy Ekstrom, Qiang Liu, Erwan Beauchamp, Lynne Postovit, Jean Wang, John R. Mackey, Naveen Pemmaraju, Gautam Borthakur, Joseph Brandwein, Luc Berthiaume. Targeting N-myristoylation for therapy of adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5662.

Abstract 3168: Immunopeptidome-defined acute myeloid leukemia progenitor cell-associated antigens are targeted in vivo by AML patients’ T cells

Abstract Despite recent advances and the approval of novel molecular therapies in acute myeloid leukemia (AML), the disease is still characterized by high relapse rates and poor overall survival due to the persistence of therapy-resistant residual leukemic progenitor cells (LPCs). T cell-based immunotherapy has been suggested as a novel therapeutic option to eliminate minimal residual disease and achieve long-lasting remissions. One main prerequisite for immunotherapy development is the selection of immunogenic targets that show natural, high-frequent, and tumor-exclusive presentation on the cell surface of malignant cells. In a previous study we characterized the antigenic landscape of AML by mass spectrometry to identify AML-specific T cell epitopes (Berlin et al. Leukemia 2015). Here, we aimed to analyze the immunopeptidome of primary AML progenitor cells (n = 10, purity >80% CD34+CD38-) to identify LPC-associated antigens that enable the specific targeting of AML LPCs. Additionally, we analyzed an extended set of AML patient samples (n = 47) to screen for naturally presented neoepitopes and to identify broadly applicable AML-associated target antigens that are presented on both AML blasts and LPCs. We identified more than 16,000 HLA class I- and 17,000 HLA class II-presented peptides on LPCs and 72,000 HLA class I and 61,000 HLA class II peptides in the total AML cohort. Comparative profiling of LPCs, AML blasts, and a benign tissue database (n = 332) revealed HLA class I- and HLA class II-presented LPC-exclusive as well as frequently presented AML-associated antigens on both AML blasts and LPCs. Besides these tumor-exclusive self-peptides, we detected naturally presented neoepitopes derived from two frequent mutations (NPM1 and IDH2) in this low-mutational burden malignancy. For immunogenicity analyses we selected 16 HLA class I- and 15 HLA class II-restricted peptides comprising unmutated as well as mutation-derived antigens. In vitro priming experiments showed the induction of peptide-specific, multi-functional, and cytotoxic CD8+ effector cells in samples of healthy volunteers and AML patients. We were able to detect strong preexisting memory T cell responses targeting LPC-associated antigens in AML patients with detection frequencies of up to 20%. Retrospective analyses revealed that patients with preexisting peptide-specific T cell responses showed improved overall survival compared to patients without any memory responses against our targets. Taken together, we identified novel, naturally presented, LPC-exclusive, and AML-associated self-antigens and neoepitopes presented on both AML blasts and LPCs. We could demonstrate the immunogenicity of 14/16 (88%) HLA class I and 13/15 (87%) HLA class II antigens highlighting their potential as promising targets for T cell-based immunotherapy approaches to eliminate minimal residual disease in AML patients. Citation Format: Annika Nelde, Heiko Schuster, Tatjana Bilich, Jens Bauer, Malte Roerden, Sarah Schroeder, Jonas S. Heitmann, Elke Rücker-Braun, Hans-Georg Rammensee, Helmut R. Salih, Juliane S. Walz. Immunopeptidome-defined acute myeloid leukemia progenitor cell-associated antigens are targeted in vivo by AML patients’ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3168.

Abstract LB102: Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia

Abstract Despite the unprecedented clinical success of autologous chimeric antigen receptor (CAR) T cell therapy in B cell leukemia and lymphoma patients, CAR T cell therapy for acute myeloid leukemia (AML) patients has lagged, in part due to the time required for autologous CAR T cell preparation in the face of the rapid relapse of AML following remission, and the possibility of on-target off-tumor hematopoietic toxicity. In this study we investigated the functional activity of an allogeneic, off-the-shelf FLT3 CAR natural killer (NK) cells expressing soluble (s) IL-15 (FLT3 CAR_s15 NK) to treat FLT3+ AML. Cord blood NK cells were first transduced with FLT3 CAR intracellular signaling constructs containing either CD28/CD3ζ or 2B4/CD3ζ. Each displayed similar cytotoxicity against FLT3+ AML in vitro (p < 0.05) and in vivo (p < 0.01) when compared to mock transduced NK cells. Cord blood NK cells transduced with either soluble (s) IL-15 (s15 NK cells) or membrane (m) bound IL-15 showed comparable extended survival in vivo and superior to NK cells transduced with sIL-15/IL-15Rα (p < 0.01) or mIL-15/IL-15Rα (p < 0.01). However, only those NK cells transduced with soluble IL-15 were able to activate neighboring non-transduced (NT) NK cells and T cells in a paracrine fashion. The FLT3 CAR_s15 NK cells were expanded ex-vivo to over 1500-fold in 16 days with ~50% of the NK cells expressing both the FLT3 CAR and sIL-15. These FLT3 CAR_s15 NK cells were then subjected to a cytotoxicity assay against the FLT3+ AML cell line (MOLM-13) and were 10 times more potent than NT NK cells (p < 0.01) and 1.7 times more potent than s15 NK cells (p < 0.05). Likewise, when co-cultured with the FLT3+ AML cell line, FLT3 CAR_s15 NK cells produced a greater amount of IFN-γ compared to NT NK cells (p < 0.01) or compared to s15 NK cells (p < 0.05). Furthermore, following cryopreservation, our thawed FLT3 CAR_s15 NK cells demonstrated high recovery (~90%) and viability (> 85%), maintaining their FLT3 CAR expression at ~50% with a phenotype and cytotoxicity that was nearly equivalent to fresh FLT3 CAR_s15 NK cells. Armed with these data we assessed the efficacy of our allogeneic, off-the-shelf FLT3 CAR_s15 NK cells in vivo using immunodeficient mice engrafted with the MOLM-13 FLT3+ cell line. Repeated infusions of FLT3 CAR_s15 NK cells provided an improved median survival compared to placebo group (36 vs 24 days; p < 0.001) or compared to identical infusions of s15 NK cells (36 vs 31 days; p < 0.05) with similar results obtained in a patient-derived xenograft model. Finally, to assess toxicity against normal peripheral blood mononuclear cells (PBMC), including FLT3+ dendritic cells, and against hematopoietic stem cells (HSCs) we performed in vitro and in vivo studies, respectively, using our FLT3 CAR_s15 NK cells. We did not observe an untoward cytotoxicity of PBMC in vitro when compared to NT or s15 NK cells and no disruption of HSC differentiation in vivo when compared to placebo group. In summary, our in vitro and in vivo studies with allogeneic off-the-shelf cord blood derived FLT3 CAR_s15 NK cells demonstrated enhanced cytotoxicity and IFN-γ secretion against FLT3+ AML in vitro and enhanced the survival of mice engrafted with FLT3+ AML without any evidence of PBMC or HSC toxicity. We believe these data collectively support a rationale for investigating this novel form of allogeneic, off-the-shelf FLT3 CAR_s15 NK cell therapy in patients stricken with FLT3+ AML. Citation Format: Anthony G. Mansour, Kun-Yu Teng, Zhiyao Li, Zheng Zu, Hanyu Chen, Aliya Ali, Jianying Zhang, Ting Lu, Shoubao Ma, Michael A. Caligiuri, Jianhua Yu. Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB102.

Abstract 3269: Dual targeting of FLT3 and LSD1 disrupts the MYC super-enhancer complex in acute myeloid leukemia

Abstract Clinical responses to kinase inhibitor therapy in acute myeloid leukemia (AML) are limited by development of resistance. A major contributor of resistance is epigenetic adaptation to kinase inhibitor therapy. We present evidence that inhibition of the epigenetic regulator lysine-specific demethylase 1 (LSD1) augments the response to inhibitors of FMS-like tyrosine kinase 3 (FLT3) in FLT3-mutant AML. We demonstrate that combined FLT3 and LSD1 inhibition results in synergistic cell death of FLT3-mutant AML cell lines and primary patient samples. High-resolution epigenetic sequencing revealed that the combination therapy synergistically suppresses pro-proliferative MYC-bound promoters and activates pro-differentiative PU.1-bound enhancers. Regulon enrichment analysis in primary AML samples nominated STAT5 (a downstream target of activated FLT3 signaling) as a putative regulator of MYC gene expression. STAT5 is highly bound to the MYC blood super-enhancer and FLT3 inhibition results in a loss of both STAT5 binding and MYC blood super-enhancer chromatin accessibility. In contrast, LSD1 inhibition suppresses MYC target genes by accumulation of repressive H3K9me1 marks. We validated these findings in 72 primary AML samples, including 19 FLT3-ITD positive AML samples. The combination improved responses in the vast majority of patient samples, and high MYC regulon activity was a predictor of response. Gene expression profiling in treated primary AML samples confirmed that dual FLT3 and LSD1 inhibition activates PU.1 target genes and suppresses MYC target genes. Finally, single cell ATAC-seq on primary AML blasts treated ex vivo with combined FLT3 and LSD1 shifted cells from a MYC super-enhancer high to MYC super-enhancer low state. Collectively, these data provide preclinical rationale for the investigation of dual FLT3 and LSD1 inhibition in clinical trial. Citation Format: William M. Yashar, Brittany M. Smith, Jake VanCampen, Garth L. Kong, Jommel Macaraeg, Daniel J. Coleman, Brian J. Druker, Julia E. Maxson, Theodore P. Braun. Dual targeting of FLT3 and LSD1 disrupts the MYC super-enhancer complex in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3269.

Caspase-2 is a mediator of apoptotic signaling in response to gemtuzumab ozogamicin in acute myeloid leukemia

The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg®) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.

Body mass index and venetoclax-hypomethylating agent induction therapy for acute myeloid leukemia.

e19038 Background: Frontline Ven-HMA dosing is imperfectly estimated with HMA using body surface area (BSA) and Ven not BSA/weight-based. Not much is known if elevated body-mass-index (BMI) reduces drug exposure and impacts clinical outcomes with Ven-HMA. We sought to investigate the effectiveness of Ven-HMA in pts with elevated BMI and hypothesized no negative impact on outcomes. Methods: We retrospectively reviewed the records of pts with AML treated with frontline Ven-HMA from 12/2018-11/2021. The primary outcome was composite response rate (CR/CRi) as defined by 2017 ELN criteria. Pearson’s χ2 tests and Wilcoxon rank tests were used for bivariate analyses. Kaplan-Meier methods were used to calculate OS. Results: We identified 63 pts treated with frontline Ven-HMA, among whom 20 had a normal BMI (<25 kg/m2), 27 were overweight (BMI 25-29 kg/m2), and 16 were obese (BMI>30 kg/m2). Median age was 73 (range 37-89) and there were no differences in baseline patient- and disease-specific factors. The cohort overall was enriched for complex karyotype and TP53-mutated disease (46% and 33%, respectively), but without intergroup differences. Increasing BMI was associated with more HMA naivety and appeared to be associated with more antifungal use during induction (BMI <25: 60%, BMI >30: 88%; p=0.07) with similar rates of moderate/strong CYP3A4 inhibition. Early mortality (within 8-weeks of Ven-HMA), was greater in obese pts, but not significantly so (BMI<25: 20%; BMI>30: 31.3%; p=0.45). CR/CRi rate was similar among groups (BMI<25: 40%; BMI 25-29: 52%; BMI>30: 44%; p=0.71), as was ORR ( p=0.14). A sub-analysis excluding pts with prior HMA did not affect the parity in rate of CR/CRi in normal weight or obese pts (40% vs 44%, p=0.83) (Table). Conclusions: Our study showed that increasing BMI appears to have no negative influence on response to frontline Ven-HMA. However, it remains unclear whether increased BMI affects drug exposure, so dedicated studies are needed.[Table: see text]

Beyond gemtuzumab ozogamicin: A systematic review of antibody therapies for acute myeloid leukemia.

e19006 Background: Acute myeloid leukemia (AML) is a neoplasm that is defined by the uncontrolled growth of myeloid progenitor cells. It is the most common form of acute leukemia in adults and is associated with poor outcomes in patients ≥ 75 years. Multiple antibody-drug conjugates (ADCs) are being investigating for their benefits in AML. Methods: A search was performed on PubMed, Cochrane, Scopus, Web of Science and ClinicalTrials.gov using the PRISMA guidelines. After screening 1,806 articles, a total of eight studies were included for this review. Results: Montesinos et al. gave a combination of talacotuzumab (anti-CD123 antibody) + decitabine to AML patients and reported an ORR of 27% (42/157) in the talacotuzumab + decitabine arm. Uy et al. studied flotetuzumab (anti-CD3 x anti-CD123 DART) and reported an ORR of 30% (9/30) in patients treated at the recommended phase two dose. Ho et al. noticed a CR in 26% (30/117) of patients who received a combination of vadastuximab talirine (anti-CD33 ADC) with azacytidine/decitabine. Feldman et al. observed that the ORR was higher in the lintuzumab (anti-CD33 antibody) + chemotherapy arm than control arm (36% vs 28%). Daver et al. and Vey et al. both studied lirilumab (anti-KIR antibody), but Daver et al. reported an ORR of 14% (5/32) in the lirilumab + azacytidine arm. In a separate study, Daver et al. administered nivolumab (anti-PD1 antibody) + azacytidine and noticed an ORR of 33% (23/70). Zeidan et al. reported that the durvalumab (anti-PD-L1 antibody) + azacytidine arm had a lower ORR than the control arm (31% vs 35%). A summary of the adverse events was provided below (Table). Conclusions: Antibody-based therapies are showing great promise for the treatment of AML but are associated with various adverse effects. More prospective clinical trials are needed to further assess the long-term benefits of such medications.[Table: see text]

Pediatric and young adult leukemia adoptive therapy (PLAT)-08: A phase 1 study of SC-DARIC33 in pediatric and young adults with relapsed or refractory CD33+ AML.

TPS7078 Background: The acute and late effects of acute myeloid leukemia (AML) therapy in children can be devastating, and relapse rates remain high. Targeted therapies are needed to decrease toxicity and improve cure rates. Most AML patients express CD33 on their leukemic blasts. CD33 is a viable target with CD33 targeted therapies approved for AML. CD33 targeted CAR T cell therapies are being developed, but safety and efficacy remain a challenge. Safety concerns include cytokine release syndrome, neurotoxicity and aplasia. Efficacy is limited, with antigen escape and T cell exhaustion proposed as mechanisms of treatment failure. Dimerizing Agent Regulated ImmunoReceptor Complex (DARIC) is a next-generation technology that utilizes a split receptor design separating the antigen binding and intracellular signaling subunits (Leung, 2019). These subunits remain inactive until the administration of rapamycin which allows for dimerization of the subunits, enabling T cell activation when antigen is present. Modulating CAR T cell activation may aid hematopoietic recovery, enhance CAR T cell function and persistence, and mitigate toxicity. We developed a DARIC T cell product, SC-DARIC33, that couples the DARIC technology with a lentiviral construct that incorporates the use of a novel humanized camelid nanobody for targeting the C2 splice isoform within the membrane proximal domain of CD33. Methods: Up to 18 patients will be enrolled in this phase 1 study (NCT05105152). Patients must have early first relapse of AML (≤ 6 months from diagnosis), first relapse refractory to at least one reinduction attempt, second or greater relapse, or refractory de novo AML. The first 3 patients must be ≥18 years, all patients who meet eligibility criteria must be ≤28 years. The primary objectives of this Phase I study (NCT05105152) are assessment of safety/toxicity and feasibility of manufacturing of SC-DARIC33. Secondary objectives include assessment of efficacy, on/off rate of DARIC expression, engraftment, expansion and persistence of SC-DARIC33. Also, the effect of cessation of rapamycin on activation state of SC-DARIC 33 and on hematopoietic recovery will be evaluated. Subjects receive lymphodepletion prior to a single dose of SC-DARIC33 T cells on Day 0 followed by initiation of rapamycin on Day +2 with the purpose of activating the T cells. Rapamycin continues until Day 21 and bone marrow evaluation occurs on Day 28. Subsequent cycles of rapamycin are allowed for patients who respond. The trial will implement a BOIN design with three possible dose levels (1 x 106, 5 x 106, and 10 x 106 DARIC+ cells/kg) to determine the maximum tolerated dose (MTD). The trial is open for enrollment at Seattle Children’s Hospital. Clinical trial information: NCT05105152.

Abstract A009: Longitudinal monitoring of pediatric acute myeloid leukemia using duplex sequencing of patient-specific panels reveals ultra-low frequency MRD that marks persistent carcinogenesis and complex clonal evolution

Abstract Rationale: Accurate and sensitive detection of measurable residual disease (MRD) is critical for clinical management and the development of new therapies for pediatric acute myeloid leukemia (AML). The presence of MRD is a well-established predictor of disease relapse and mortality. Ideally, MRD detection would be both broadly applicable across patients and highly specific, thus enabling clinicians to make timely treatment decisions to improve outcomes. Background: In pediatric AML, unlike adult AML, there are few cross-patient recurrent somatic driver mutations. This makes it impractical to use a fixed gene panel for MRD monitoring. One solution is to perform disease tracking with patient-specific mutations that are present in individual AMLs at time of diagnosis (TOD). However, following modern therapies, should residual disease remain, the telltale mutations will be present in only a very small subpopulation of cells–far below the ~1% limit-of-detection of conventional NGS methods. Duplex sequencing (DS) is an ultra-accurate NGS error-correction method which increases the accuracy of base calls by more than 10,000-fold. Here we demonstrate the use of tumor-informed DS to detect patient-specific MRD in pediatric AML samples with unprecedented sensitivity and specificity. Methods: Marrow aspirates were collected from 13 pediatric AML patients at TOD, during treatment, remission, and at relapse. Among the cohort, 9/13 patients ultimately relapsed. Personalized hybrid capture panels were designed to target hundreds of somatic mutations that were present in each patient’s TOD sample, as identified by whole genome sequencing of tumor-normal pairs. DS was used for longitudinal monitoring of these mutations in blood or marrow at up to 5 time points following initial induction. Results: We detected MRD in one or more samples from all 9 patients who went on to relapse. However, we also observed high-confidence TOD variants in the last collected sample from all 5 patients who did not relapse during at least 24 months of clinical follow-up. As a technical control, we tested a subset of patient panels on a blood sample from a healthy subject and found no disease-specific mutations. Interestingly, we observed a marked degree of heterogeneity of response-to-therapy in the cohort. In some cases, all mutations logarithmically decreased uniformly, whereas in others there was clear dispersion of subclones over time, with some declining 100-times more quickly than others. Conclusions: While DS in conjunction with personalized panels represents a powerful approach for MRD tracking, simply the existence of very low frequency TOD mutations in pediatric AML is not specific enough for predicting relapse without additional interpretation. Lingering mutations in non-relapsers might represent preneoplastic field defects, pre-existing somatic mosaicism, or perhaps even true residual disease under immune suppression. Our study highlights an unappreciated degree of complexity that arises when using ultra-accurate mutation detection for cancer monitoring. Citation Format: Thomas H. Smith, Jake E. Higgins, Fang Y. Lo, Michael J. Hipp, Charles C. Valentine, Amanda R. Leonti, Rhonda E. Ries, Soheil Meshinchi, Benjamin J. Huang, Jesse J. Salk. Longitudinal monitoring of pediatric acute myeloid leukemia using duplex sequencing of patient-specific panels reveals ultra-low frequency MRD that marks persistent carcinogenesis and complex clonal evolution [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A009.

Hsa-miR-181a-5p, hsa-miR-182-5p, and hsa-miR-26a-5p as potential biomarkers for BCR-ABL1 among adult chronic myeloid leukemia treated with tyrosine kinase inhibitors at the molecular response

BackgroundTyrosine kinase inhibitors (TKIs) as first-line therapy for Chronic Myeloid Leukemia (CML) show a high success rate. However, a low number of patients with long-term treatment-free remission (TFR) were observed. Molecular relapse after imatinib discontinuation occurred at 50% at 24 months, with 80% occurrence within the first 6 months. One of the reasons for relapse is untimely TKIs discontinuation caused by large errors from estimates at very low-level or undetectable disease, thus warranting new biomarkers for CML.MethodsNext Generation Sequencing (NGS) was used to identify microRNAs (miRNAs) at the molecular response in CML adult patients receiving TKIs treatment. A total of 86 samples were collected, 30 from CML patients responsive and 28 from non-responsive to imatinib therapy, and 28 from blood donors. NGS was conducted whereby 18 miRNAs were selected and validated by real-time RT-qPCR in triplicate.ResultsHsa-miR-181a-5p was expressed significantly (p-value< 0.05) with 2.14 and 2.33-fold down-regulation in both patient groups, respectively meanwhile hsa-miR-182-5p and hsa-miR-26a-5p were significant only in the non-responsive group with 2.08 and 2.39 fold up-regulation. The down-regulation was consistent with decreased amounts of BCR-ABL1 in patients taking TKIs regardless of molecular responses. The up-regulation was consistent with the substantial presence of BCR-ABL1 in CML patients treated with TKIs at the molecular response.ConclusionsTherefore, these miRNAs have potential as new therapeutic biomarkers for BCR-ABL1 status in adult CML patients treated with TKIs at molecular responses. These could improve current approaches and require further analysis to look for targets of these miRNAs in CML.

Potential to Improve Therapy of Chronic Myeloid Leukemia (CML), Especially for Patients with Older Age: Incidence, Mortality, and Survival Rates of Patients with CML in Switzerland from 1995 to 2017.

Simple SummaryIn a population-based study of chronic myeloid leukemia (CML) patients in Switzerland, we confirmed an increase in relative survival for all age groups over the last decades. This was primarily based on the stable age-adjusted rate of incidence and a substantial decrease of the age-adjusted mortality rate. Investigating data from four different study periods, before and after introduction of tyrosine kinase inhibitors and their more potent second- and third-generation compounds, we found higher increases in relative survival for older patients at later time periods compared to younger CML patients. However, for the last study period (2013–2017), the five-year relative survival (RS) in the elderly population reached only 53% compared to 89% in younger patients, implicating additional potential to improve CML therapy, especially in the elderly population.Background: Tyrosine kinase inhibitors (TKI) substantially improved chronic myeloid leukemia (CML) prognosis. We aimed to describe time period- and age-dependent outcomes by reporting real-world data of CML patients from Switzerland. Methods: Population-based incidence, mortality, and survival were assessed for four different study periods and age groups on the basis of aggregated data from Swiss Cantonal Cancer Registries. Results: A total of 1552 new CML cases were reported from 1995 to 2017. The age-standardized rate (ASR) for the incidence remained stable, while the ASR for mortality decreased by 50–80%, resulting in a five-year RS from 36% to 74% over all four age groups. Importantly, for patients <60 years (yrs), the five-year RS increased only in earlier time periods up to 92%, whereas for older patients (+80 yrs), the five-year RS continued to increase later, however, reaching only 53% until 2017. Conclusions: This is the first population-based study of CML patients in Switzerland confirming similar data compared to other population-based registries in Europe. The RS increased significantly in all age groups over the last decades after the establishment of TKI therapy. Interestingly, we found a more prominent increase in RS of patients with older age at later observation periods (45%) compared to patients at younger age (10%), implicating a greater benefit from TKI treatment for elderly occurring with delay since the establishment of TKI therapy. Our findings suggest more potential to improve CML therapy, especially for older patients.

Response to Tyrosine Kinase Inhibitors in Real-World Patients With Chronic Myeloid Leukemia.

Tyrosine kinase inhibitors (TKIs) are the front-line therapy for chronic myeloid leukemia (CML), where phase 3 clinical trials have demonstrated their safety and efficacy. However, trial patients may not be representative of real-world patients (RWPs). To evaluate RWP clinical factors associated with effectiveness and safety in CML patients treated with TKIs. Patients with CML treated with at least 30 days of imatinib, dasatinib, nilotinib, or bosutinib between 2014 and 2018 were included. Patients were stratified into categories based on the number of factors that would have precluded enrollment into pivotal TKI phase 3 trials (0, 1, ≥2). End points included complete hematologic response (CHR), early molecular response (EMR), major molecular response (MMR), adverse event (AE)-induced dose decreases, treatment interruptions, and treatment discontinuations. Final analyses included 174 patients. Patients with ≥2 factors had a higher risk of dose decreases (relative risk = 1.54; 95% CI = 1.02-2.34; P = 0.02) and a shorter time to dose decrease (hazard ratio = 2.43; 95% CI = 1.23-4.97; P = 0.006) compared with patients with 0 factors. Significant differences were observed in CHR at 1 month and MMR at 3 months between patients with 0 and ≥2 factors (P = 0.03 and P = 0.04, respectively). Approximately 60% of our RWPs would have been excluded from the pivotal phase 3 TKI trials. These data suggest that RWPs require more precise dosing to achieve CML clinical milestones and to mitigate AEs, but findings should be validated prospectively.

Abstract 938: Pharmacologic targeting Mcl-1 with AZD5991 induces apoptosis, suppresses mitochondrial respiration and overcomes ibrutinib resistance in non-Hodgkin lymphoma cells

Abstract Bcl-2 family proteins determine cell fate and comprise pro-apoptotic “initiators” (Noxa, Bim, Puma), anti-apoptotic “guardians” (Bcl-2, Mcl-1, Bcl-xL) and pro-apoptotic effectors (Bax/Bak). Venetoclax, a Bcl-2 inhibitor, received regulatory approval in the therapy of chronic lymphocytic leukemia and acute myeloid leukemia. However, venetoclax is modestly effective in non-Hodgkin lymphoma (NHL). Mcl-1 is a short-lived pro-survival protein that is frequently overexpressed in NHL, leading to increased cell survival and drug resistance. Mcl-1 transgenic mice develop aggressive lymphoma. Thus, selective targeting Mcl-1 represents a promising pharmacologic strategy in NHL. AZD5991 is a selective small-molecule inhibitor with high potency against Mcl-1 which has entered clinical trials. Here we explored its pre-clinical activity in NHL. We detected Mcl-1 expression in ten diffuse large B-cell lymphoma (DLBCL) as well as in parental/ibrutinib-resistant JeKo-1 and Mino mantle cell lymphoma (MCL) cell lines. AZD5991 suppressed proliferation and induced apoptosis in DLBCL cells in a dose-dependent manner, as well as in parental/ibrutinib-resistant MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction, including mitochondrial membrane depolarization, decreased mitochondrial mass, increased mitophagy, and increased levels of reactive oxygen species in both DLBCL and parental/ibrutinib-resistant MCL cells. Mcl-1 inhibition downmodulated basal and maximal respiratory capacity in sensitive, but not in resistant cells, as determined by the Seahorse assay, further indicating mitochondria compromise as a mechanism of susceptibility to Mcl-1 inhibition. An inhibitors screening assay identifies that AZD5991 demonstrated synergy with other BH3-mimetics. Co-treatment of DLBCL cells with Bcl-2/xL inhibitors AZD4320, venetoclax and navitoclax overcame resistance to AZD5991. Finally, Primary MCL cells were co-cultured with control, CD40L- or BAFF-expressing stroma which partially mimics the lymph node microenvironment and induces resistance to spontaneous and drug-induced apoptosis. Mcl-1 inhibition induced apoptosis of primary MCL cells in both control and BAFF-expressing conditions, accompanied by decreased mitochondrial respiration. Meanwhile, CD40L stroma rendered protection from AZD5991 which was partially overcome by co-treatment with venetoclax. In summary, Mcl-1 inhibition using selective BH3-mimetic AZD5991 restricts cell proliferation and induces apoptosis in a subset of DLBCL, parental/ibrutinib-resistant MCL cells and primary neoplastic B cells. Mcl-1 inhibition leads to mitochondrial dysfunction and inhibition of cellular respiration. Resistance to Mcl-1 inhibition may be overcome by concurrent targeting of alternative anti-apoptotic proteins (Bcl-2/xL) in NHL. Citation Format: Tingting Liu, Vi Lam, Elana Thieme, Xiaoguang Wang, Fei Xu, Steve Kurtz, Jeff Tyner, Olga V. Danilova, Alexey Danilov. Pharmacologic targeting Mcl-1 with AZD5991 induces apoptosis, suppresses mitochondrial respiration and overcomes ibrutinib resistance in non-Hodgkin lymphoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 938.

An evaluation of venetoclax in combination with azacitidine, decitabine, or low-dose cytarabine as therapy for acute myeloid leukemia

ABSTRACT Introduction Older patients with acute myeloid leukemia (AML) ineligible for conventional chemotherapy have historically received low-intensity treatments, if any, and have had dismal outcomes. Recent phase III data have demonstrated significant efficacy of venetoclax-based combinations and have begun to address the unmet need in this patient population. As venetoclax-based combinations become increasingly used in the clinical setting, it is important to understand their development, current use, and future directions. Areas covered This review covers the clinical development of venetoclax-based combinations for the management of AML, and their current and future use. A search of PubMed and ashpublications.org using the keywords ‘venetoclax’, ‘AML’, and ‘hypomethylating agents’ as the search terms was undertaken to identify the most pertinent publications. Expert opinion While venetoclax-based combinations have shown excellent responses and improved survival in patients with untreated AML, further studies are required to understand how to expand on their frontline use, manage patients who fail venetoclax-based combinations, and their true efficacy in the relapsed/refractory setting. Management of AML with venetoclax-based combinations is expected to evolve over the next few years.

Low Plasma Citrate Levels and Specific Transcriptional Signatures Associated with Quiescence of CD34+ Progenitors Predict Azacitidine Therapy Failure in MDS/AML Patients.

Simple SummaryEpigenetic drugs, such as azacitidine (AZA), hold promise in the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), however, the mechanisms predicting the patients’ response to AZA is not completely understood. Quiescence of hematopoietic CD34+ progenitors has been proposed as a predictive factor for AZA therapy failure in MDS/AML patients, but the interplay between CD34+ cell cycle status and their metabolic signature in a predisposition to AZA (non)responsiveness remains unclear. Our data on patients with MDS or AML with myelodysplasia-related changes (AML-MRC) suggest that AZA-responders have actively cycling CD34+ cells poised for erythro-myeloid differentiation, with high metabolic activity controlling histone acetylation. Conversely, the patients who progressed early on AZA therapy revealed quiescence signature of their CD34+ cells, with signs of reduced metabolically-controlled acetylation of histones needed for transcription-permissive chromatin configuration. Our study delineates plasma citrate levels and CD34+ cells’ transcriptional signatures associated with cycling status and metabolic characteristics as factors predicting the response to AZA monotherapy in MDS/AML-MRC patients.To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients’ HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.

Downregulation of MTSS1 in acute myeloid leukemia is associated with a poor prognosis, chemotherapy resistance, and disease aggressiveness

Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML.

Proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), play an oncogenic role in chronic myeloid leukemia by stabilizing nuclear factor-kappa B

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have revolutionized therapy for chronic myeloid leukemia (CML), paving the way for clinical development in other diseases. Despite success, targeting leukemic stem cells and overcoming drug resistance remain challenges for curative cancer therapy. To identify drivers of kinase-independent TKI resistance in CML, we performed genome-wide expression analyses on TKI-resistant versus sensitive CML cell lines, revealing a nuclear factor-kappa B (NF-κB) expression signature. Nucleocytoplasmic fractionation and luciferase reporter assays confirmed increased NF-κB activity in the nucleus of TKI-resistant versus sensitive CML cell lines and CD34+ patient samples. Two genes that were upregulated in TKI-resistant CML cells were proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), both members of the 19S regulatory complex in the 26S proteasome. PSMD1 and PSMD3 were also identified as survival-critical genes in a published small hairpin RNA library screen of TKI resistance. We observed markedly higher levels of PSMD1 and PSMD3 mRNA in CML patients who had progressed to the blast phase compared with the chronic phase of the disease. Knockdown of PSMD1 or PSMD3 protein correlated with reduced survival and increased apoptosis in CML cells, but not in normal cord blood CD34+ progenitors. Luciferase reporter assays and immunoblot analyses demonstrated that PSMD1 and PSMD3 promote NF-κB protein expression in CML, and that signal transducer and activator of transcription 3 (STAT3) further activates NF-κB in scenarios of TKI resistance. Our data identify NF-κB as a transcriptional driver in TKI resistance, and implicate PSMD1 and PSMD3 as plausible therapeutic targets worthy of future investigation in CML and possibly other malignancies.

Oral therapies for acute myeloid leukemia in 2021: a team work! The Toulouse center experience

Oral therapies for acute myeloid leukemia in 2021: a team work! The Toulouse center experience

Efficacy and cost-benefit of filgrastim administered after early assessment bone marrow biopsy during induction therapy for acute myeloid leukemia

The role of filgrastim during acute myeloid leukemia (AML) induction therapy remains controversial. At our institution, newly diagnosed AML patients from 2003 through 2019 were retrospectively evaluated. Patients were stratified on whether they received filgrastim within 5 days after early assessment bone marrow (BMBx) and divided into early GCSF group (eGCSF) and no-eGCSF group. A total of 121 patients were included. We found significantly shorter hospital stay (median 24 vs 26 days, p < .01), absolute neutrophil count recovery days (median 23 vs 25 days, p = .03), and intravenous antibiotics days (mean 18.5 vs 21.4 days, p = .01) in patients with eGCSF comparing with no-eGCSF. There was no significant difference regarding complete response rates; however, for those failed to achieve remission, eGCSF was associated with higher blast count. There was no significant difference regarding overall survival or progression-free survival. The use of eGCSF was associated with cost savings of $5199 per patient over no-eGCSF.