Cusatuzumab is a monoclonal antibody that binds with high affinity to CD70, a cell surface protein overexpressed on CD34+ acute myeloid leukemia (AML) progenitors and leukemia stem cells. This phase Ib study assessed the safety, tolerability and efficacy of adding cusatuzumab to standard-of-care azacitidine and venetoclax (CVA) or a cusatuzumab and venetoclax (CV) doublet regimen in patients newly diagnosed with AML who were ineligible for intensive chemotherapy. Cusatuzumab 20 mg/kg was administered intravenously on days 3 and 17 of 28-day cycles combined with standard dosing of venetoclax ± azacitidine. Overall, 44 patients were treated with CVA. Common hematologic treatment-emergent adverse events (TEAEs) were neutropenia (77.3%), thrombocytopenia (77.3%) and anemia (45.5%); non-hematologic TEAEs included nausea (45.5%), diarrhea (43.2%), constipation (40.9%), fatigue (36.4%) and vomiting (31.8%). Grade ≥3 infectious complications included febrile neutropenia (38.6%), sepsis (36.4%) and pneumonia (9.1%). The overall composite response rate (complete remission [CR] + CR with partial hematologic recovery [CRh] + CR with incomplete hematologic recovery [CRi]) was 77.3% (CR, 47.7%; CRh, 20.5%; CRi, 9.1%). Among CR/CRi responders, 53% achieved negative measurable residual disease by multiparameter flow cytometry. Median overall survival was 12.0 months (95% confidence interval: 8.7-NE) months. Sixteen patients were treated with CV and had similar safety but less favorable responses and survival than those treated with CVA. These results support further development of CVA for the treatment of AML (clinicaltrials.gov identifier: NCT04150887).

Despite advances in acute myeloid leukemia (AML) treatment, significant unmet medical needs remain. Surrogate end points for overall survival can accelerate the approval of novel therapies. Measurable residual disease (MRD) is a promising surrogate end point candidate, providing a sensitive and quantitative assessment of disease burden. Numerous studies have demonstrated that negative MRD status-across diverse methodologies, assessment timepoints and thresholds, patient subgroups, and clinical settings-independently predicts improved survival. Although MRD can inform therapeutic decisions at the patient level, its formal integration as a primary surrogate end point in regulatory frameworks for clinical trials requires rigorous validation. MRD Partnership and Alliance in AML Clinical Treatment (MPAACT) is a research consortium among industry and academic leaders. MPAACT actively engages with regulatory agencies, health technology assessment bodies, technology vendors, and patient groups to establish a pathway for validating MRD as a surrogate end point in AML clinical trials. For the current article, the authors reviewed the status of MRD assessment, its use in recent clinical trials, current MRD assessment methodologies, standardization, regulatory guidance, statistical approaches, and patient access considerations necessary for MRD to become a surrogate clinical trial end point. The extensive collaboration between MPAACT, global industry, and academic partners-including data sharing and resource integration-underscores the collective commitment to advancing AML therapies. Establishing MRD as a surrogate end point could accelerate the development and approval of innovative treatments, ultimately improving patient outcomes.

PURPOSE The objective of this study was to develop a flexible risk stratification strategy for AML that is specific for venetoclax plus azacitidine (ven/aza), addresses real-world data (RWD) issues, and is also adaptable to different use cases. METHODS A series of tunable risk models (RMs) were generated from a dynamic counterfactual machine learning (ML) strategy. These used a range of features from diagnostic AML samples and were tested using objective metrics on a single-institution cohort of 316 newly diagnosed patients treated with ven/aza. RM performance was tested using various model assumptions, data elements, and end points and with applications to an external AML real-world cohort (RWC). RESULTS Favorable, intermediate, and adverse risk groups were identified in a series of ML-based RMs using different assumptions, for genetic-only or genetic-plus-phenotypic data elements and with overall survival and event-free survival as end points. Most RMs demonstrated equitable patient distribution (approximately 20%-40% in each risk group), significant separation between risk strata (log-rank-based P values <0.001), and predictability computed by time-dependent survival AUC values of 0.60-0.70. Similar performance was observed when the proposed RM strategy was adapted and compared with the European Leukemia Net 2022 using the external RWC. CONCLUSION The proposed ML strategy addresses a variety of RWD considerations and is readily tunable through coding and parameter updates for different contexts and use case needs. This strategy represents a novel approach to developing more effective RMs for AML and possibly other diseases.

Background and Significance: Acute myeloid leukemia (AML) is an aggressive, biologically heterogeneous hematopoietic stem cell neoplasm with historically dismal outcomes in patients unable to receive intensive chemotherapy. Based on the pivotal phase 3 VIALE-A study (DiNardo et. al. NEJM, 2020), the combination of venetoclax (Ven), a selective oral BCL-2 inhibitor, with azacitidine (Aza) was approved for the treatment of newly diagnosed AML in adults 75 years or older, or adults ³18 years with comorbidities precluding intensive induction chemotherapy. Overall survival (OS) in VIALE-A was 14.7 months for Ven-Aza vs 9.6 months for placebo-Aza. Among patients with a poor cytogenetic risk the median OS was 7.6 months. Cusatuzumab (Cusa) is a monoclonal antibody with high affinity to human CD70, a cell surface protein expressed on a variety of cancers with limited expression in normal tissues other than activated immune cells. Cusa blocks CD70/CD27 signaling, leading to inhibition of leukemia stem cell proliferation and reduction in leukemic blast cells and exerts direct Fc-mediated, effector functions such as enhanced antibody-dependent cellular cytotoxicity. A Phase 1/2 study (NCT03030612) and a Phase 2 study (NCT04023526) combining Cusa and Aza were conducted in patients with AML unable to tolerate intensive chemotherapy. The combination was generally well-tolerated, with most treatment-emergent adverse events consistent with those expected for hypomethylating agents and there was no obvious dose dependency for toxicities. Manageable infusion-related reactions related to Cusa were noted (Pabst et al. Haematologica, 2023). Subsequently, a single arm Phase 1b study of Cusa combined with Ven and Aza (VAC) for patients with AML was performed (NCT04150887). CR for VAC was 47.6%, CR + CRh + CRi 81.0% and MLFS 11.9% in 42 evaluable patients. Among responders, 47% were MRD negative (Roboz et al, Blood 2021, 138: 369). Overall, the study results showed promising efficacy and safety and support the further development of VAC induction for newly diagnosed patients with AML. Study Design and Methods: OV-AML-1231 is a randomized, open-label, multicenter, multinational, Phase 2 trial to evaluate the efficacy and safety of VAC compared to VA in patients with newly diagnosed AML ineligible for intensive chemotherapy (NCT06384261) that started accrual in July 2024. The planned enrollment is 120 newly diagnosed AML patients, randomized 2:1 to VAC (80 patients) and VA (40 patients). Participants will receive standard of care VA or Cusa 20 mg/kg administered by intravenous infusion on Day 3 and Day 17 of 28-day cycles in combination with VA. The trial population will be enriched for patients with adverse cytogenetic and molecular risk features. The primary end point is OS. Secondary endpoints include event-free survival, response rates including CR, CR+CRh, and rates of MRD negativity. Planned exploratory studies include defining differential responses of AML genetically subgroups to the two treatment arms. Key eligibility criteria include age of 18 years or older and a confirmed diagnosis of previously untreated AML. Eligible patients will include those 75 years of age or older or patients with at least one of the following coexisting conditions: ECOG performance status of 2 or 3, history of congestive heart failure requiring treatment or ejection fraction ≤50% or chronic stable angina, diffusion capacity of the lungs for carbon monoxide (DLCO) ≤65% or forced expiratory volume in the first second (FEV1) ≤65%. Patients with prior exposure to hypomethylating agents are excluded. This ongoing, multicenter, randomized study may inform new frontline low-intensity therapeutic options for newly diagnosed AML patients with high-risk biological features.

Acute myeloid leukemia (AML) in older patients has a poor prognosis, low complete remission (CR) rates, and poor overall survival (OS). Preclinical studies have shown synergistic effects of epigenetic priming with hypomethylating agents followed by cytarabine. Based on these data, we hypothesized that an induction regimen using epigenetic priming with decitabine, followed by cytarabine would be effective and safe in older patients with previously untreated AML. Here, we conducted a phase 2 trial in which older patients with previously untreated AML received an induction regimen consisting of 1 or 2 courses of decitabine 20 mg/m2 intravenously (IV) for 5 days followed by cytarabine 100 mg/m2 continuous IV infusion for 5 days. Forty-four patients (median age 76 years) were enrolled, and CR/CRi was achieved by 26 patients (59% of all patients, 66.7% of evaluable patients). Fourteen of 21 (66.7%) patients with adverse cytogenetics achieved CR including six out of seven evaluable patients with TP53 mutations. The 4- and 8-week mortality rates were 2.3% and 9.1%, respectively, with median OS of 10.7 months. These results suggest epigenetic priming with decitabine followed by cytarabine should be considered as an option for first-line therapy in older patients with AML. This trial was registered at www.clinicaltrials.gov as # NCT01829503.

Venetoclax plus azacitidine (ven/aza) is a new standard of care for adult Acute Myeloid Leukemia (AML) patients who are not candidates for intensive therapies. Risk stratification approaches have been proposed to identify patients with favorable, intermediate, and adverse therapeutic outcomes following ven/aza and other lower intensive therapies. However, most have been developed for retrospective data analyses and have limitations in their application to upfront risk stratification of newly diagnosed patients. Here, we describe an AML risk model, termed the Refined Risk Model (RRM), that is specific for ven/aza, addresses important real-world considerations and utilizes pathology features that have the potential to be available relatively quickly-and-broadly following diagnosis. The RRM was developed and internally validated using a single center cohort of 316 AML patients from the University of Colorado treated upfront with ven/aza, and then externally validated on an AML cohort from a nationwide electronic health record-derived de-identified AML database. The RRM effectively stratified patients into Adverse, Intermediate, and Favorable groups across both the internal and external cohorts; it performed well in subsets with or without allogeneic transplant recipients, demonstrated tolerance to missing data, and showed numerical performance comparable to or exceeding the existing alternatives such as the European Leukemia Network (ELN 2022) and molecular prognostic risk signature (mPRS) models. These findings suggest that the RRM may have potential application in defining the prognostic mortality risk for newly diagnosed AML patients, which may help guide clinical trial design and execution as well as other important elements of AML clinical decision support. ### Competing Interest Statement Both CAS and MB are employees of and hold equity in OncoVerity. In addition, CAS is a consultant to RefinedScience. All other authors declare no conflicts of interest. ### Funding Statement This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was a retrospective analysis utilizing a limited data set. Study approval and a full HIPAA waiver of consent was granted by the Colorado Multiple Institutional Review Board (approval number 23-2059) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes This retrospective study was approved by CU internal review board (IRB) and used a limited dataset with a waiver of consent from the CU IRB. The raw, individual patient data are protected and not available due to data privacy laws. The processed data are available at reasonable request to the corresponding author. The Flatiron Health data that supported the findings of this study were originated by and are the property of Flatiron Health, Inc., which has restrictions prohibiting the authors from making the data set publicly available. Requests for data sharing by license or by permission for the specific purpose of replicating results in this manuscript can be submitted to PublicationsDataAccess@flatiron.com.

ABSTRACT:Enasidenib (ENA) is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML substudy, we applied a risk-adapted approach to assess the efficacy of ENA monotherapy for patients aged ≥60 years with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated that mutant IDH2 was in the dominant leukemic clone. Patients for whom ENA monotherapy did not induce a complete remission (CR) or CR with incomplete blood count recovery (CRi) enrolled in a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to ENA alone demonstrated efficacy, with a composite complete response (cCR) rate (CR/CRi) of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and 1 dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of ENA monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML. This trial is registered at www.clinicaltrials.gov as #NCT03013998.

Objective The objective of this study was to develop a flexible risk model (RM) stratification strategy for Acute Myeloid Leukemia (AML) that is specific for the new standard of care venetoclax plus azacitidine (ven/aza), captures disease heterogeneity, and addresses a range of real-world data issues. Materials and Methods A series of tunable RMs based on a dynamic counterfactual machine learning (ML) strategy that utilized next generation sequencing, cytogenetics, flow cytometry, and other features of the diagnostic AML samples were developed and tested on a single institutional cohort of 316 newly diagnosed patients treated initially with ven/aza. Results Favorable, Intermediate, and Adverse risk groups were identified in a series of novel RMs derived using ML models for overall survival (OS) and event free survival (EFS). Most, but not all models, demonstrated equitable patient distribution into the different risk categories (∼20%-40% in each group) with significant separation between categories (Log-Rank based p-values <0.001), and with predictability computed by survival AUC values in the ∼0.60-0.70 range. Discussion and Conclusion The general strategy employed here is specific for AML patients treated with ven/aza, is based on a wide range of diagnostic AML pathology features, considers feature interactions, addresses data missingness, sparsity, and the confounding effects of allogeneic hematopoietic cell transplant. It is also readily tunable through simple coding and context specific parameter updates, and adaptable to reflect different use case needs. This strategy represents a new approach to developing more effective RMs for AML and possibly other diseases as well. ### Competing Interest Statement Both CAS and MB are employees of and hold equity in OncoVerity. In addition, CAS is a consultant to RefinedScience. All other authors declare no conflicts of interest. ### Funding Statement This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was a retrospective analysis utilizing a limited data set. A full waiver of consent and a full waiver of Health Insurance Portability and Accountability Act (HIPAA) authorization were granted by the Colorado Multiple Institutional Review Board (approval number 23-2059). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes

Supplementary Figure 1 from Triggering of Toll-like Receptor 4 Expressed on Human Head and Neck Squamous Cell Carcinoma Promotes Tumor Development and Protects the Tumor from Immune Attack

Figure S1.ABBV-744 exhibits potent anti-proliferative activity against AML cells through cell cycle arrest and induction of apoptosis.

Figure S6.Identification of biomarker of response to ABBV-744/venetoclax therapy.

Figure S4.ABBV-744 displaces BRD4 from regulatory regions of BCL2, BCL2L1 and RUNX1 in sensitive AML cells but not insensitive AML line.

Konstantinos Lontos , Anastasia Tsagianni, Mounzer Agha, Anastasios Raptis, Jing-Zhou Hou, Rafic Farah, Robert L. Redner, Annie Im, Kathleen A. Dorritie, Alison Sehgal , James Rossetti, Nidhi Aggarwal, Melissa Saul, Michael Boyiadzis and Nathanael G. Bailey UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA; Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

Supplemental Table 3. Multivariate analysis showing impact of cGVHD characteristics on treatment related mortality, disease free survival and overall survival in patients with CML who developed cGVHD

Primary patient sample information

Supplemental Table 1. Multivariate analysis of late relapse, transplant-related mortality, overall survival after HCT for each disease (AML, ALL, CML, MDS)

Figure S2. ABBV-744 retains strong transcription regulatory activities in AML cells and it did not alter the baseline expression of representative IFN-r or PMA responsive genes.

Background Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes, thus there remains a need to integrate molecular information to identify patients most likely to respond to drugs and combinations. Disease segmentation based on transcriptomics has provided valuable insight into disease risk and the likelihood of response to targeted compounds. IL2RA(CD25) is a receptor expressed on both AML leukemic and immune cells, and has shown initial promise as a potential dual target of leukemic blasts and regulatory T (Treg) cells in pre-clinical models (Pousse et al. Front. Oncol. 2023). We used a systems approach based on a recently described transcriptomic classifier for AML (Hamidi et al. ASH 2021) and other molecular tools to characterize the association between IL2RA expression levels and AML genomic markers, clinical features and patient outcomes. In addition, we evaluated IL2RA in relation to ex vivo drug sensitivity, to identify patient segments who are most likely to benefit from CD25-targeting drugs. Methods BEAT-AML (NCT01728402) RNAseq data from patient samples, associated ex vivo drug sensitivity data (N=283), was VOOM normalized. Patients were binned into quartiles based on IL2RA expression. Gene signature scoring was performed using xCell cell type enrichment algorithm and GSVA for Hallmark pathways and scRNAseq signature based on Van Galen et al. Cell 2019. Associations were performed with clinical outcome (log-rank test), gene signatures (Spearman correlation), mutations (Wilcoxon test) and ex vivo drug sensitivity (Kruskal Wallis test). Results We characterized the BeatAML dataset and found that elevated IL2RA expression associates with inferior overall survival (p=0.014) and high-risk features. In addition, we identified a strong association between IL2RA expression and FLT3-ITD status (Figure), as well as other genetic alterations. Using correlation analyses, we established an association between IL2RA expression levels and “primitive” AML signatures (leukemic stem cell (LSC), R=0.4, p=6.8x10-12, hematopoietic stem cell (HSC)-like, R=0.43, p=6.8x10-14) and Tregs (R=0.47, p=1.7x10-15), and an anti-correlation with promonocytic signatures (R=-0.23, p=1.1x10-4). Interestingly, HSC-like and Treg signatures were also correlated (R=0.19, p=0.00087), consistent with an association between stem cell abundance and a repressive immune microenvironment. Finally, using a multivariate model adjusting for the effect of Tregs and LSCs, the prognostic value of IL2RA remained significant (p=0.029). We previously used unsupervised machine learning clustering based on consensus non-negative factorization (cNMF) to discover novel transcription-based classification (Hamidi ASH 2021). Using this methodology, we identified a strong correlation between IL2RA expression in cNMF subtypes (p=1.6x10-10). Moreover, this method uncovered patient subtypes in which IL2RA correlates with LSC (cNMF 6.3), Treg (6.4, 6.6) or both signatures (6.1, 6.2). This differential association was independent of prognostic category, maturation state or venetoclax sensitivity (Table). Transcription-based classifiers have been shown to be highly predictive of ex vivo drug sensitivity. Consistent with an association between IL2RA expression and FLT3-ITD status, we identified strong correlations between IL2RA and sensitivity to FLT-3 and other tyrosine kinase inhibitors, providing a rationale for combining these compounds with CD25-targeting therapeutics. In contrast, there was no correlation between IL2RA levels and venetoclax AUC (p=0.25), while the cNMF classification system similarly revealed that patient segments with increased IL2RA expression had heterogeneous venetoclax sensitivity (Table), supporting CD25 targeting in patients who may not respond to venetoclax. Conclusion AML patients with elevated IL2RA expression have inferior prognosis, enrichment of stem-like and Treg signatures and FLT3-ITD alterations. Transcriptomics-based clustering could be used to guide combination therapies based on potential impact of CD25 targeting on Tregs, leukemic cells, or both populations. Ex vivo drug sensitivity analyses support combinations of CD25-targeting agents with FLT3 inhibitors, as well as venetoclax. Additional work to evaluate these and other combinations using functional assays is warranted.

Supplementary figure and table legends