Efficacy of the Peptide Drug Conjugates Melflufen and OPDC3 in Venetoclax Resistant Acute Myeloid Leukemia
Authors: Kumari Romika , Miettinen Juho J. , Tambe Mahesh B. , Olgac June , Ruokoranta Tanja , Ikonen Nemo , Suvela Minna H. , Huppunen Maiju-Emilia , Svensson Gelius Stefan , Acs Klara , Heckman Caroline A. 
Published: December 2024

Background: The combination of BCL2 inhibitor venetoclax with a hypomethylating agent (azacitidine or decitabine) or low dose cytarabine has transformed acute myeloid leukemia (AML) treatment, especially for older patients who are unfit for intensive chemotherapy. However, the relatively short duration of response and development of resistance by different cellular mechanisms remains a major concern (Saliba, John et al. 2021)-(Ong, Kim et al. 2022). Better understanding of resistance mechanisms and new therapeutic options are urgently needed for patients relapsing or refractory to venetoclax-based treatment. Melflufen and OPDC3 are peptide-drug conjugates (PDCs) that utilize elevated peptidase and esterase expression in cancer cells to rapidly and selectively release cytotoxic agents inside the cells (Wickström, Malin et al. 2017; Miettinen, J. Juho et al. 2021). Melflufen was recently approved by European Medical Agency for relapse/refractory multiple myeloma. Here we evaluated the activity of two PDCs, melflufen and OPDC3, in relapsed or refractory AML.
Aims: To discover new treatment options especially for venetoclax resistant AML, we assessed the efficacy of venetoclax and the PDCs melflufen and OPDC3 in samples from patients with relapsed/refractory AML and in AML cell models of acquired venetoclax resistance. Further, we applied single cell RNA-sequencing (scRNA-seq) to identify cellular and genomic biomarkers of drug sensitivity and resistance.
Methods: In total, 20 viably frozen bone marrow mononuclear cell samples from patients with AML taken at the relapse/refractory stage, were received from the Finnish Hematology Registry and Clinical Biobank (FHRB). The FHRB collected the samples after informed consent and using approved protocols in accordance with the Declaration of Helsinki. High-throughput flow cytometry (HT-FC)-based ex vivo drug sensitivity testing was performed using the iQue Screener Plus flow cytometer and ForeCyt software. Cells were incubated with drugs for 3 days and the impact measured using CD34, CD117, CD14, and CD45 antibodies, Annexin V and 7AAD. Drug sensitivities of the detected cell populations were assessed using a drug sensitivity score (DSS) (Yadav, Pemovska et al. 2014). Drug sensitivity testing of the AML cell lines with acquired resistance to venetoclax was performed using CellTiter Glow assay. The AML cell lines were HL-60, KASUMI-1, MOLM-13, and MV4-11.
For scRNA-seq, libraries were prepared using the 10x Genomics Chromium platform and then sequenced with the Illumina NovaSeq 6000 system. 10x Genomics Cell Ranger v6.0.2 pipelines were used for data processing and analysis. The downstream analysis was performed using the Seurat tool and cell-type annotations were done using gene markers from sctype (Ianevski, Giri et al. 2022) and multimodal reference mapping (Hao, Hao et al. 2021).
Results: Our patient cohort consisted of 11 male and 9 female patients with a median age of 62 years and represented different FAB sub-types (FAB M1 (n=5); FAB M2 (n=5); FAB M4 (n=2); FAB M5 (n=4), or no FAB information (n=4)), as well as distinct karyotype profiles. The median blast cell percentage was 49% (minimum=25%; maximum=90%). HT-FC analysis of BM-MNCs revealed comparable drug sensitivity to melflufen and OPDC3, whereas venetoclax exhibited a distinct drug sensitivity profile among the tested samples. Blast cells in FAB M5 patient samples were clearly more sensitive to OPDC3 and melflufen compared to venetoclax. Regardless of FAB sub-type, patient samples that were resistant to venetoclax exhibited good sensitivity to melflufen and OPDC3. This observation was further validated in AML cell lines with acquired resistance to venetoclax. By scRNA-seq analysis, a total of 17 different cell populations were identified including different myeloid and lymphoid cell-types. For OPDC3, the proportion of the HSC/prog like cells, HSC/MPP cells and neutrophils in the samples showed significant association with the drug response.
Conclusion: Based on ex vivo, functional assessment, the peptide drug conjugates melflufen and OPDC3 are highly active in AML, especially in models with primary or acquired resistance to venetoclax and support further investigations of these drugs in venetoclax-resistant AML.