We assessed the addition of the tyrosine kinase inhibitor Quizartinib, following intensive chemotherapy and as maintenance, in patients aged >60 years with AML or high-risk MDS, regardless of FLT3 mutation status. 463 patients (median age 68yrs) were randomised (1:1) to receive Quizartinib 40mg or not for 14 days immediately following chemotherapy courses 2 and 3, plus 28 additional days; those allocated Quizartinib were further randomised (1:1) to either 12 additional 28-day maintenance courses (long Quizartinib), or no further treatment (short Quizartinib). Median follow-up was 76 months. 314 patients were FLT3 wild type (WT); 116 had FLT3 mutations. The primary endpoint, overall survival (OS) unselected by FLT3 status, showed no significant difference (HR 0.99, 95% CI 0.79-1.24, p=0.937) and there was an increase in non-relapse mortality with Quizartinib (HR 1.64, 95% CI 1.04-2.59, p=0.032). In a pre-planned subgroup analysis, FLT3-mutated patients who received Quizartinib had significantly improved OS (HR 0.59, 95% CI 0.37-0.93, p=0.024) due to reduced relapse risk (HR 0.57, 95% CI 0.35-0.91, p=0.017) with greater benefit in the short Quizartinib group (HR 0.49, 95% CI 0.24-1.02, p=0.055). In FLT3-WT patients there was no survival benefit and no reduction in relapse risk. No significant differences were seen in time to hematologic count recovery or in the duration of hospitalisation. The most observed grade 3/4 adverse events were febrile neutropenia. In conclusion, the addition of Quizartinib to intensive chemotherapy, delayed until chemotherapy course 2, prolonged OS in older patients with FLT3-mutated AML but did not improve OS in non-FLT3 selected patients. ISRCTN-31682779, EudraCR-2013-002730-21.

We compared daunorubicin/AraC plus fractionated gemtuzumab (DAGO2) with CPX-351 (CPX) (1:2 randomisation) in 439 patients ≥60yrs (median age 68yrs) without known adverse-risk cytogenetics entering the NCRI AML18 version2 trial (NCT02272478). Median follow-up was 35 months. Patients not in MRD-negative remission after course-1 could enter a second randomization between standard versus intensified chemotherapy. Post course-1 response rates were greater after DAGO2 (CR+CRi, 60% vs 47.5%, OR 0.61 95%CI 0.41-0.91, p=0.016). Following course-2 the overall response was not significantly different, 85% for DAGO2 vs 78% for CPX (OR 0.64, 95%CI 0.39-1.09, P=0.095). More patients attained CR with MRD negativity post course-1 in the DAGO2 arm (47% vs 29% for CPX, OR 0.46 95%CI 0.29-0.72, p=0.004). We observed better 3yr EFS (34% vs 27%, HR 0.73 95%CI 0.57-0.93, P=0.012) and OS (52% vs 35%, HR 0.62, 95%CI 0.46-0.83, P=0.001) with DAGO2. In a stratified analysis, CPX did not provide a survival benefit in patients with MDS-related mutations (HR 1.40, 95%CI 0.97-2.03) and was associated with poorer survival in patients with NPM1 (HR 2.83 95%CI 1.17-6.82) and FLT3 mutations (HR 2.14, 95%CI 0.98-4.68). 37% of patients were transplanted in CR1 and this did not differ by randomization. Survival post-transplant did not differ between arms. For patients entering the course-2 randomisation (n=107) survival was equivalent between standard versus intensified CPX doses (P=0.565).In this population of older patients without known adverse-risk cytogenetics, DAGO2 resulted in superior survival compared to CPX. CPX did not benefit those with MDS-related mutations over DAGO2.

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies characterized by the accumulation of clonal, abnormally differentiated blasts in the bone marrow. For most patients, prognosis remains poor and a large proportion relapse. Using a small cohort of AML blasts, we previously showed using proteomic analysis that the expression of the transcription factor, nuclear factor 1C (NFIC) was elevated in 69% (9/13) of AML patients compared to normal hematopoietic stem and progenitor cells (HSPC). Further, that ectopic expression of NFIC in HSPC promoted growth and monocytic development and that NFIC knockdown (KD) reduced the growth of AML blasts. Here we show the clinical significance of NFIC expression, and the molecular mechanisms and cellular pathways utilized by NFIC to promote AML growth. We first determined NFIC protein expression in a larger, more heterogeneous group of AML blasts obtained from the UK-MRC AML15 clinical trial. We analyzed the protein composition of 91 primary AML blasts and compared NFIC expression to normal human CD34+ HSPC, CD14+ monocyte and CD15+ granulocyte controls using mass spectrometry. Consistent with our previous data, NFIC protein expression was detected in 68% (62/91) of AML blasts and undetectable in normal blood cell populations, a result confirmed by western blotting. To determine the clinical significance of NFIC expression, we initially analyzed AML patient blast mRNA expression data using the BEAT AML dataset. Comparing European Leukemia Network prognosis groups, NFIC mRNA expression was significantly higher among AML patients with favorable prognosis compared to intermediate and adverse groups. NFIC expression was strongest among core-binding factor AML (CBFB::MYH11 and RUNX1::RUNX1T1) and acute promyelocytic leukemia. The presence of NPM1 or FLT3-ITD mutations had no clinically significant effect on NFIC expression. NFIC expression did not correlate with patient age, gender, white blood cell count or French-American-British grouping suggesting elevated NFIC expression is an independent prognostic factor. To identify the mechanisms utilized by NFIC to support AML cell growth, we analyzed NFIC's effects on gene expression and chromatin organization. The effect of NFIC KD was analyzed in THP-1 cells using mRNA sequencing coupled with promotor capture Hi-C (PCHi-C) sequencing and compared to scrambled control (SCR). Enrichment analysis of differentially expressed genes (log2FC≥1) revealed that NFIC KD in AML cells promoted expression of genes involved in nucleosome assembly and chromatin silencing. Meanwhile, NFIC KD suppressed expression of genes involved in extracellular matrix (ECM) organization, PI3K-Akt pathway signaling and amino acid synthesis, transport and metabolism. PCHi-C sequencing identified 192 differential chromatin interactions involving 256 unique gene promotors. To identify the upstream functional targets of NFIC, we interrogated our list of ~1800 differentially expressed genes, obtained by mRNA sequencing, for genes with differential promotor interactions. This yielded 16 potential functional targets of NFIC in AML cells, including ADAM28, which has previously been shown to regulate the growth, migration and invasion of AML cells. To determine which of these targets showed correlative expression with NFIC in AML patients, we carried out a Pearson correlation analysis of mRNA data from the TARGET AML dataset. Of the 16 genes, 4 (PCOLCE2, SAMD10, SHMT2 and ZNF512B) correlated with NFIC expression in AML patients (R2 > 0.3). Finally, we validated differential expression of SHMT2 and ADAM28 (included due to its previous association with AML) in NFIC KD THP-1 cells compared to SCR using western blotting. No difference in PCOLCE2 expression was detected and expression of SAMD10 and ZNF512B were undetectable in all samples using western blotting. In summary we show that NFIC overexpression in AML cells primarily induces PI3K-Akt pathway signaling, amino acid metabolism and ECM reorganization to support AML growth providing new potential avenues for therapeutic targeting of NFIC signaling in AML. Further, we identify SHMT2 and ADAM28 as potential upstream targets of NFIC that may be key in mediating NFIC's pro-growth effects in AML and are currently determining whether ectopic expression of these proteins can rescue the effect of NFIC knock down.

Introduction Despite repeated vaccinations against SARS-CoV-2 virus, patients who are immunocompromised remain at very high risk of catching SARS-CoV-2 virus and becoming unwell. AZD7442 (Evusheld) is a long-acting monoclonal antibody treatment that has been shown in clinical trials to prevent SARS-CoV-2 infection for up to a year after a single dose. Vaccines require a healthy immune system to generate protective immunity. AZD7442 may prevent SARS-CoV-2 infection in immunocompromised individuals that may not have responded to repeated vaccinations against SARS-CoV-2 virus. Unlike vaccinations, AZD7442 reaches effective levels within the body a few hours after a single dose. The RAPID-PROTECTION trial will determine the levels of immune protection that AZD7442 offers patients at the very highest risk of SARS-CoV-2 infection and whether this protection can be further enhanced by repeated vaccination against SARS-CoV-2 virus.Methods RAPID-PROTECTION is a multicentre, interventional and open-label adaptive platform trial that aims to recruit 350 immunocompromised participants across five UK centres. Participants will be administered AZD7442 on day 0 followed by a SARS-CoV-2 vaccination 28 days later. Participants will be randomised (1:1) to the Moderna vaccine or Pfizer/BioNTech vaccine. Participant samples will be taken at baseline and at multiple timepoints after the administration of AZD7442.Analysis The participant samples will be analysed to measure the function and magnitude of SARS-CoV-2 specific antibody and T-cell responses at baseline and at multiple timepoints after the administration of AZD7442. The immunological effect of the study interventions will be determined by comparison of the results of immunological assessments at baseline and subsequent timepoints.Ethics and dissemination The trial protocol was approved by the research ethics committee of the National Health Service (reference 22/HRA/0359), Health Research Authority and Health and Care Research Wales on 25 July 2022. Findings will be disseminated through peer-reviewed journals and presented at scientific conferences.Trial registration number ISRCTN53507177.

We compared daunorubicin/AraC plus fractionated gemtuzumab (DAGO2) with CPX-351 (CPX) (1:2 randomisation) in 439 patients ≥60yrs (median age 68yrs) without known adverse-risk cytogenetics entering the NCRI AML18 version2 trial (NCT02272478). Median follow-up was 35 months. Patients not in MRD-negative remission after course-1 could enter a second randomization between standard versus intensified chemotherapy. Post course-1 response rates were greater after DAGO2 (CR+CRi, 60% vs 47.5%, OR 0.61 95%CI 0.41-0.91, p=0.016). Following course-2 the overall response was not significantly different, 85% for DAGO2 vs 78% for CPX (OR 0.64, 95%CI 0.39-1.09, P=0.095). More patients attained CR with MRD negativity post course-1 in the DAGO2 arm (47% vs 29% for CPX, OR 0.46 95%CI 0.29-0.72, p=0.004). We observed better 3yr EFS (34% vs 27%, HR 0.73 95%CI 0.57-0.93, P=0.012) and OS (52% vs 35%, HR 0.62, 95%CI 0.46-0.83, P=0.001) with DAGO2. In a stratified analysis, CPX did not provide a survival benefit in patients with MDS-related mutations (HR 1.40, 95%CI 0.97-2.03) and was associated with poorer survival in patients with NPM1 (HR 2.83 95%CI 1.17-6.82) and FLT3 mutations (HR 2.14, 95%CI 0.98-4.68). 37% of patients were transplanted in CR1 and this did not differ by randomization. Survival post-transplant did not differ between arms. For patients entering the course-2 randomisation (n=107) survival was equivalent between standard versus intensified CPX doses (P=0.565).In this population of older patients without known adverse-risk cytogenetics, DAGO2 resulted in superior survival compared to CPX. CPX did not benefit those with MDS-related mutations over DAGO2.

Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q ('chr. 21amp') in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A, a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target.

Most patients with acute myeloid leukemia (AML) may obtain remission upon induction chemotherapy, but relapse is frequent and associated with poor survival. Previous prognostic models for outcomes after relapse lacked analysis of comprehensive molecular data. A validated prognostic model integrating clinical, cytogenetic, and molecular variables may support treatment decisions. We studied 943 patients with AML who relapsed after intensive induction treatment in a development cohort (HOVON-SAKK). A random survival forest algorithm was used to evaluate the association of clinical parameters, cytogenetic abnormalities, and molecular variables at diagnosis with overall survival (OS). Relapsing patients (n = 377) who were enrolled in the NCRI-AML18 trial were used for validation. In the development cohort, the median age at relapse was 58 years, and patients were classified as 2022 European LeukemiaNet favorable (22%), intermediate (31%), and adverse risk (48%). One-third underwent allogeneic transplantation in the first complete remission. Variable selection yielded 9 variables associated with 1-year OS, including relapse-free interval, age, white blood cell count, mutated TP53, FLT3 internal tandem duplication, core-binding factor abnormalities, t(v;11q23)/KMT2A rearrangement, and complex/monosomal karyotype, which were assigned points according to their estimated hazard ratios. Three prognostic groups were defined with distinct 1-year OS in both development (favorable, 51% ± 3%; intermediate, 29% ± 3%; and poor, 14% ± 2%, respectively) and validation cohorts (51% ± 4%, 26% ± 5%, and 14% ± 3%, respectively). Validation confirmed the improved accuracy in predicting outcomes for patients with AML in first relapse. The revised AML relapse model improved on previous prognostic models for outcomes after first relapse. It provides stratification that might support tailoring second line treatment.

BACKGROUND:In patients with acute myeloid leukaemia treated with curative intent, the detection of measurable residual disease (MRD) generally confers a poor prognosis. This study aimed to identify whether altering treatment based on MRD results can improve survival. METHODS:In the UK NCRI AML17 and AML19 randomised, controlled, phase 3 trials, performed in the UK, Denmark, and New Zealand, we screened patients aged 16-60 years with newly diagnosed acute myeloid leukaemia for molecular markers suitable for disease monitoring, including NPM1 mutations and fusion genes. Patients with a marker were randomly assigned (2:1) to either sequential molecular MRD monitoring during treatment and for 3 years after, or standard clinical care only with no molecular monitoring. In the monitoring group, treating physicians decided whether and how to incorporate the MRD results into the patient's therapy, including in cases of MRD relapse. The primary endpoint was overall survival. Prespecified subgroup analysis of the primary outcome included analysis by molecular group (NPM1mut with FLT3-ITD, NPM1mut without FLT3-ITD, and fusion gene transcripts). Both trials were registered with ISRCTN, ISRCTN55675535 and ISRCTN78449203, and are completed. FINDINGS:In the AML17 trial, 1836 patients were enrolled between June 1, 2012 and Dec 31, 2014. In the AML19 trial, 965 patients were enrolled between Nov 9, 2015, and Jan 23, 2018. 637 patients were randomly assigned across both trials (289 to MRD monitoring and 144 to no monitoring in AML17 and 136 to MRD monitoring and 68 to no monitoring in AML19). With a median follow-up time of 4·9 years (IQR 3·6-5·9), overall survival at 3 years was 70% (95% CI 66-75) in patients in the monitoring group and 73% (68-80) in patients in the no-monitoring group. Meta analysis of the two studies showed no difference in overall survival (hazard ratio [HR] 1·11, 95% CI 0·83-1·49; p=0·25). In the pre-specified subgroup analysis of the primary endpoint, overall survival at 3 years in patients with both NPM1 and FLT3 internal tandem duplication (ITD) mutations was 69% (95% CI 60-79) in the monitoring group and 58% (45-74) in the no-monitoring group (HR 0·53, 95% CI 0·31-0·91; p=0·021). However there was no difference in survival by randomisation in patients with NPM1 mutations without FLT3-ITD (overall survial 69% [95% CI 62-77] in the monitoring group and 78% [70-87] in the no monitoring group; HR 1·56, 95% CI 0·96-2·52) or those with fusion gene transcripts (overall survial 72% [95% CI 65-79] in the monitoring group and 77% [68-87] in the no monitoring group; HR 1·28, 95% CI 0·80-2·18). INTERPRETATION:Sequential molecular MRD monitoring, coupled with MRD-guided treatment, did not improve overall survival in the entire study population; however, in the subgroup of patients with baseline NPM1 and FLT3 ITD mutations, we observed a survival benefit for MRD monitoring. FUNDING:National Institute for Health Research, Blood Cancer UK, and Cancer Research UK.

Introduction Acute myeloid leukaemia (AML) is a potentially fatal haematological malignancy characterized by the uncontrolled growth of leukemic cells (myeloid blasts), causing bone marrow failure. Despite recent advances in targeted therapeutics, high mortality rates persist across all age groups particularly the patients above the age of 60 years, emphasizing the need for novel and less toxic therapeutic strategies. KAT2A is a histone acetyltransferase involved in transcriptional regulation as part of two multisubunit complexes: Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC), modulating lineage specificity and metabolic/cell-cycle pathways respectively. KAT2A has been shown to be upregulated in AML, where it plays a pivotal role in leukemic stem cell maintenance and chemotherapy resistance. Pharmacological inhibition of KAT2A has been shown to trigger differentiation in AML blasts while largely sparing normal haematopoietic progenitors, indicating that KAT2A blockade is a mechanistically selective therapeutic approach. We investigated the efficacy and mechanism of action of a novel proteolysis-targeting chimera (PROTAC) degrader; MDI0117218 which targets KAT2A as a differentiation-inducing therapeutic strategy in primary AML blasts. Methods AML cell lines (MOLM-13, KG1a, OCI-AML3, NB4, HL60) and a large cohort (n=50) of primary AML samples were assessed across a range of assays including: ATP-based proliferation assays (Cell-titre Glo), cytospin morphology, PKH26 cell division tracking and flow cytometric evaluation of differentiation-associated surface markers. KAT2A degradation and subsequent acetylation reduction was monitored by Western blot and intracellular flow for sub-population analysis. Long-term co-culture efficacy with HS5 stroma and colony assays were established to evaluate PROTAC effects on drug resistant adherent and putative stem cell fractions. Synergistic interaction with standard of care agents Cytarabine, Venetoclax and Azacitidine were assessed in combination and pretreatment-washout experiments. Results MDI0117218 showed nanomolar efficacy in AML cell lines (MOLM13 and OCI-AML3) and 48% of primary AML samples (n=50, Average EC50 33nM +/-324nM in sensitive primary AML samples, Ave 6.6uM +/- 1.4uM in resistant primary AML samples). This effect was observed across both differentiated and undifferentiated FAB types of AML. Western blotting in primary AML samples confirmed potent and rapid target engagement, with complete and sustained KAT2A degradation observed from 5nM within 30 minutes of incubation. Intracellular flow cytometry confirmed uniform reduction of KAT2A across blast sub-populations in sensitive patient samples compared to those with resistant EC50s. H3K9 acetylation reduction also correlated with KAT2A knockdown in sensitive samples providing potential clinical biomarker readouts. Assessment of MDI0117218 mechanism of action revealed variable cytostatic and differentiation responses with induction of CD11b +/- terminal CD14 differentiation in responders accompanied by cell cycle arrest without apoptotic induction consistent with myeloid differentiation which was confirmed by morphological analysis. Drug efficacy and differentiation was sustained in longer-term stromal co-culture and Colony forming assays, with significant reduction in all blast sub-populations including stromal adherent drug-resistant blasts and colony forming units (p<0.02, n=10). Combination drug assays in ex vivo primary AML blasts showed moderate to additive synergistic interaction between MDI0117218 and cytarabine (Average CI at ED50 0.9 +/- 0.85) and good synergy with Venetoclax (Average CI at ED50 of 0.25 +/- 0.09) across a range of doses. Pre-treatment and sequential washout protocols confirmed synergy to standard agents highlighting the rapid and sustained knockdown effects of MDI0117218. Conclusion MDI0117218 is an effective and rapid KAT2A degrader across multiple models of cell line and primary AML. Differentiation and anti-proliferative effects were observed across a wide range of differentiated and non-differentiated AML patients, highlighting the potential for a broad acting agent with potential for combination with both Cytarabine and Venetoclax; two mainstays of treatment for transplant-ineligible or relapsed AML. Taken together with favourable pre-clinical toxicology profiles, these data support the rationale for this agent as a strong candidate for early-phase clinical trials.

Background Inobrodib (CCS1477) is a first in class potent, selective, and orally bioavailable inhibitor of the bromodomains of p300 and CBP, two closely related histone acetyltransferases with oncogenic roles in hematological malignancies. Inobrodib exhibits potent anti-tumor cell activity in a range of hematological cell lines, including multiple myeloma, and demonstrates additive/synergistic activity with pomalidomide. Aims: We report preliminary safety (primary objective) and efficacy (secondary) data for inobrodib in combination with pomalidomide (pom) and dexamethasone (dex) from relapsed/refractory multiple myeloma (RRMM) patients treated in the Phase I/IIa trial (NCT04068597). Methods : Eligible patients (pts) had confirmed RRMM and had exhausted available or suitable standard of care treatment options. Two dose escalation combination cohorts were completed; pts received inobrodib at doses of 25 mg or 35 mg bidaily on a 4 days on/3 days off intermittent schedule, as well as standard dosing of pom and dex in 28-day cycles. Adverse events were graded by CTCAE v5.0. Responses were investigator assessed per IMWG. Results: Initial dose escalation cohorts enrolled 15 RRMM pts with a median age of 71 yrs (range 41-80). Median prior lines of therapy was 5 (range 3-9). Median follow-up was 56 days (range 13-189), with a median number of 3 cycles received (range 1-7). Almost all patients were pomalidomide-refractory (13/15, 87%), 13/13 (100%, 2 pts data missing) were triple class refractory and 3 pts (20%) received prior BCMA therapy. At the data cut-off (5 th June 2023), Grade (gr) 3 /4 treatment-emergent adverse events (TEAEs) were reported in 11 of 15 (73%) pts. Over half of the patients remain on treatment and all but three remain in follow-up for survival analysis. Of these three patients, two died due to disease progression and one due to an unrelated cardiac event at the end of cycle 2. The most frequent gr 3/4 events were infections (41.1%). The majority of the other gr 3/4 TEAE were hematological and seen in 5 of 13 (38.5%) pts; neutropenia (23.5%) thrombocytopenia (5.9%, all gr 3) and anemia (11.8%, all gr 3). Seven of 15 (47%) pts experienced TEAEs considered related to inobrodib; all events were gr 1/2 apart from a single patient with gr 3 electrolyte abnormalities. No DLTs were noted; two pts (13%) discontinued due to related events. The majority of patients 13/15 (87%) showed signs of activity with disease markers decreasing over time. At the point of data cut off, 7/15 (47%) heavily pre-treated patients achieved confirmed responses (MR or better) per IMWG criteria; 2 VGPR, 3 PR and 2 MR. Of note, two further patients, who initially received inobrodib monotherapy but progressed were transferred into the pom-dex combination cohort and subsequently achieved objective responses (VGPR and PR). Summary: The first in class agent inobrodib in combination with pom-dex shows promising early efficacy in heavily pre-treated, including pom-refractory, RRMM patients. The combination is tolerable, with the most common events being infections, as expected in this patient population. Dose optimisation continues as part of this study with expansion arms in an earlier setting planned.

ABSTRACT:Despite the use of FMS-like tyrosine kinase 3 (FLT3) inhibitors, outcomes for patients with FLT3-mutated (FLT3mut) acute myeloid leukemia (AML) remain suboptimal because of high rates of relapse. We evaluated the safety and efficacy of the combination of daunorubicin, cytarabine (DA), gemtuzumab ozogamicin (GO), and midostaurin (DAGO+m) for younger patients with newly diagnosed FLT3mut AML in the UK National Cancer Research Institute AML19 trial. A total of 195 patients were randomized to receive DA with either 1 or 2 doses of GO (DAGO1 and DAGO2). Overall, 77 had an FLT3 mutation and received midostaurin for 2 weeks after each chemotherapy course and then as maintenance for 1 year unless they received a transplant. A total of 39 patients received DAGO1+m and 38 DAGO2+m. Their median age was 51 years (range, 20-74), and 16 (20%) were aged >60 years. The overall response rate was 91%. Day 60 mortality was 0%, with no increase in toxicity compared with patients treated contemporaneously with DAGO1 and DAGO2 without midostaurin. Two-year overall survival was 77%. Two-year event-free survival and cumulative incidence of relapse were 62% and 31%, respectively. Measurable residual disease (MRD) clearance was enhanced compared with patients with FLT3mut AML treated with DAGO without midostaurin. Overall, 81% of evaluable patients were NPM1 MRD negative in the peripheral blood after course 2 (76% with DAGO1+m, and 86% with DAGO2+m), 79% were MRD negative in the bone marrow by FLT3-ITD next-generation sequencing, and all patients had FLT3-MRD levels <0.01%. DAGO+m appears safe and effective. DAGO2+m will now be evaluated in a randomized study. This trial was registered at www.isrctn.com as #ISRCTN78449203.

Despite advances in targeted therapies, the 5-year survival rate for Acute myeloid leukemia (AML) remains poor, particularly for patients over 60 years of age, underscoring the urgent need for novel therapeutic approaches. Our group's previous comparison of the nuclear proteome and transcriptome of AML primary blasts with normal CD34+ identified several differentially expressed proteins, including S100A4. This calcium-binding protein was significantly overexpressed (5.5-fold) in the nuclei of 11 out of 15 AML patients compared to cord blood derived CD34+ cells (Alanazi et al, Leukemia 2020). S100A4 is known to regulate various biological processes including cell cycle progression, proliferation, and differentiation. It has been studied in the context of solid tumor metastasis. However, its potential involvement in hematological malignancies is an emerging area of research, and the underlying mechanisms of its contribution to AML development and progression are not well understood. We demonstrate that S100A4 is essential for AML cell proliferation and survival through a series of in vitro experiments. Knockdown (KD) of S100A4 using targeted shRNA significantly impaired the growth of AML cell lines, reducing growth by 7-fold (p<0.0001). Flow cytometry analysis revealed that S100A4 KD induced cell cycle arrest, with a 28.9% ± 2.1% increase in G1 phase cells and decrease in S and G2 phases cells compared to control cells (p<0.0001). Additionally, Annexin V/PI staining showed a 2.7-fold increase in early apoptotic cells and a 2.1-fold increase in late apoptotic/necrotic cells in S100A4-depleted cells (p<0.0001). These findings were validated in 5 primary AML samples, where S100A4 KD reduced growth and survival of AML patient blasts by an average of 3.6-fold (p<0.0001). RNA-sequencing revealed substantial alterations in gene expression profiles following S100A4 KD in AML cell lines (THP-1, TF-1, NOMO-1, OCI-AML2). We identified 3,850 upregulated and 3,205 downregulated genes (fold change>1.5, adjusted p-value <0.05), with enrichment in processes related to inflammation, immune response, cell cycle regulation, apoptosis, and autophagy. Downregulation of S100A4 was associated with a 2.3-fold upregulation of NCOA7 across various AML cells (p<0.001). The mRNA expression data from an AML patient cohort (Ley et al. 2013) revealed a significant negative correlation between S100A4 and NCOA7 expression levels (-0.42, p=2.59×10−8). This is consistent with our observation that S100A4 KD leads to increased NCOA7 expression in AML cells. Our study revealed a novel S100A4/NCOA7/V-ATPase axis in AML, where S100A4 KD led to 5-fold upregulation of NCOA7 protein expression (p<0.001). Further investigation into the functional relationship between NCOA7 and S100A4 revealed that NCOA7 CRISPR-cas9 rescued the survival defect in AML cells with S100A4 KD. V-ATPase activity is essential for maintaining normal intracellular pH homeostasis. Acidification of the cytoplasm is a characteristic feature of cells undergoing apoptosis. Intracellular pH measurements verified that V-ATPase activity was increased in S100A4 KD cells, leading to a decrease in intracellular pH. Pharmacological inhibition of V-ATPase in the S100A4-depleted cells using bafilomycin A and concanamycin A, lead to an increase in intracellular pH mirroring the rescue effect and suggesting a mechanism by which S100A4 promotes AML cell survival through downregulation of NCOA7 and activation of V-ATPase. Western blot analysis showed increased expression of V-ATPase subunits ATP6V1B2 (4.5-fold) and ATP6V0A1 (3.4-fold) in S100A4 KD cells (p < 0.001). Furthermore, we investigated the role of S100A4 in modulating the response of AML cells to chemotherapeutic agents. Overexpression of S100A4 in MV4-11 cells resulted in resistance to Cytarabine and Daunorubicin (p<0.001). In contrast, S100A4 overexpression in KG-1 cells led to increased sensitivity to Cytarabine and Daunorubicin (p<0.0001), highlighting the cell line-specific effects of S100A4 on chemotherapy response. These findings suggest that the impact of S100A4 expression on chemotherapy efficacy may depend on the molecular and cytogenetic background of the AML cells. Our study reveals a novel S100A4/NCOA7/V-ATPase axis in AML and suggests that targeting S100A4 may improve treatment outcomes by addressing drug resistance and enhancing therapeutic efficacy.

Background Notable advances have been made in the treatment of patients with myeloid malignancies in recent years, thereby making health-related quality of life (HRQoL) a critical aspect to consider. However, limited data exists on the HRQoL and symptom burden profile of these patients. Objective The main objective of this study was to assess the prevalence of clinically important symptoms in a large cohort of newly diagnosed patients with AML, APL and MDS. A secondary objective was to investigate differences in the baseline HRQoL profile across these diseases. Methods: Data were gathered through the PROACTIVE project, a large international initiative aimed at enhancing evidence-based knowledge on patient-reported outcomes (PRO) in patients with AML/MDS. To this end, a number of high-quality datasets (including at least the EORTC QLQ-C30) from international cooperative groups are being pulled together. For the purpose of this study, a sample of 2370 patients from NCRI-UK trials and 1551 newly diagnosed patients from GIMEMA studies were combined, resulting in a final sample of 3921 patients. Only patients with valid baseline (pretreatment) HRQoL questionnaires were considered for this analysis (n=3349/3921, 85%). A total of 2046 (61%) patients were diagnosed with AML with a median age of 69 years, 1078 (32%) diagnosed with MDS, with a median age of 73 years; the remaining 225 patients (7%) were diagnosed with APL with a median age of 46 years. The prevalence of clinically important symptoms in the overall population and by disease group was based on previously established thresholds for the EORTC QLQ-C30 (Giesinger JM, et al.J Clin Epidemiol. 2020 Feb;118:1-8). A higher score for a functional scale represents a higher level of functioning while a high score for a symptom scale represents a higher level of symptomatology. Multivariable linear regression models were used to assess cross-sectional mean differences between types of diagnosis for the EORTC QLQ-C30 scales. Given the higher prevalence of patients with AML, we used this as a reference category for each regression model adjusted for sex and age. The magnitude of clinical relevance (small, medium and large) of the adjusted mean differences was evaluated using established scale-specific thresholds. Results Analysis of baseline symptom prevalence in the overall population indicated a substantial burden of the disease with the 5 most prevalent clinically important symptoms being: dyspnea (69%), nausea/vomiting (58%), pain (48%), fatigue (40%) and appetite loss (24%). No statistically significant difference was observed in the prevalence of fatigue across AML, APL and MDS patients. However, the prevalence of other symptoms varied by disease, with APL patients typically reporting higher prevalence followed by AML and MDS. For example, nausea/vomiting was 89%, 70% and 30% (p<.001) for APL, AML and MDS respectively. Also, pain was 60%, 53% and 34% (p<.001) for APL, AML and MDS respectively. Investigation of the baseline HRQoL profile revealed differences across diseases. Relative to patients with AML, those with APL had worse large clinically relevant differences for cognitive functioning (∆ = -23.7), nausea/vomiting (∆ = 24.8), and dyspnea (∆ = 17.5). APL patients also had worse medium to small clinically relevant differences for appetite loss (∆ = 14.3), role functioning (∆ = -9.9), constipation (∆ = 9.7), pain (∆ = 9.4), diarrhea (∆ = 7.9), physical functioning (∆ = -6.0), insomnia (∆ = 5.1) and global QoL (∆ = -4.4). Relative to patients with AML, those with MDS reported large better clinically relevant differences regarding dyspnea (∆ = -18.3) and nausea/vomiting (∆ = -25.5). MDS patients also had medium to small better clinically relevant differences for cognitive functioning (∆ = 11.9), global QoL (∆ = 6.2) as well as appetite loss (∆ = -13.3) and pain (∆ = -12.8). Conclusion: Our findings indicate that patients with AML, APL and MDS experience a high symptom burden already at the time of diagnosis. Current data also helps to characterize the specific baseline HRQoL profile of these patients highlighting most relevant functional aspects and symptoms in need of special attention for each disease. Future analyses are planned to investigate longitudinal trajectories of HRQoL by type of treatment and other key characteristics

Introduction: The AML19 v2 ‘midotarg’ pilot explored the safety and efficacy of Midostaurin in patients with a FLT3 mutation who had received DA chemotherapy combined with one or two doses of Gemtuzumab Ozogamicin (GO) in induction. The addition of Midostaurin to DAGO1 and DAGO2 chemotherapy was well tolerated and safe in both younger and older patients with FLT3mut AML with promising survival at 24 months. The full clinical outcomes of this study have been reported (Russell N, ASH 2023), and here we report the findings from the patient reported outcome measures (PROs) across treatment groups. Methods: Patients with newly-diagnosed AML without known adverse karyotype were randomised to receive DA 3+10 plus either a single dose of GO or two doses. Patients with a FLT3-ITD or TKD could enter the “Midotarg” pilot and receive 50mg bd of Midostaurin (m) for 14 days following completion of chemotherapy, and following the second induction (DA 3+8 without GO) and 2 courses of HDAC consolidation and as maintenance for 12 cycles in non-transplanted patients. Quality of Life (QoL) questionnaires were given to all new patients prior to starting treatment, prior to course 2 of treatment, and at 3, 6 (approximately post consolidation), 9 and 12 months post trial entry. The questionnaires included the European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC QLQC-30). Scores were calculated following the EORTC scoring manual reporting mean values across domains. Data was analysed using a linear mixed model to compare scores across treatment groups. A more stringent p-value (0.0025) was set for this exploratory analysis to account for the multiplicity of testing. Consistent with current evidence a minimal clinically important difference (MCID) of ≥10 was set to measure deterioration across time points, using a logistic regression analysis to estimate the odds ratio of MCID. Results: 196 patients with a median age of 50 (IQR 40-58) years were recruited to AML19 v2 between November 2020 to November 2021, of which 77 FLT3mut patients were enrolled into the Midotarg pilot receiving DAGO1m (n=39) or DAGO2m (n=38). 59 had a FLT3 ITD and 22 a FLT3-TKD (and 4 had both). The remaining, predominantly FLT3 WT, patients (n=119) received DAGO1 (n=59) or DAG02 (n=60) only. Of these patients, 161 (82%) provided baseline PRO data, with completion of 120 (61%) at post course 2, 114 (58%) at 3 months, 105 (54%) at 6 months, 97 (49%) at 9 months and 91 (46%) at 12 months. There were no statistically significant differences in global QOL between the DAGOm and DAGO treatment groups. Both groups followed the same trajectory of improvement, with an increase in global QOL scores from baseline to prior to course 2 (47 to 71 DAGOm, 54 to 71 DAGO) which was maintained through to 12 months. A significant treatment time interaction was observed on the individual domains for dyspnea at 6 months (Avg score DAGOm 19 (SE3.9), DAGO 32 (SE4.4); P = 0.001) and for insomnia at 6 months (Avg score DAGOm 21(SE4.3), DAGO 34 (SE4.8); P = 0.002) and 9 months (Avg score DAGOm 19 (SE3.4), DAGO 33 (SE5.0); P = 0.002). At 6 and 9 months DAGOm patients were on maintenance midostaurin whereas DAGO patients were off treatment which may account for this effect. At 12 months all patients were off treatment and there was no statistical difference across domains between treatment groups. No further statistically significant differences were observed between the treatment groups across any other scales. Additionally, there were no significant differences in the proportion of patients experiencing clinically meaningful deterioration at any time point between treatment groups. Conclusion: The analysis of the PROs from this pilot study demonstrate that the promising clinical benefits previously reported are not accompanied by any clinically relevant adverse deterioration in patient's overall QOL. This was an exploratory analysis that offers important insight for hypothesis generation for future studies in this patient group.