AML Blasts Intrinsic Interferon Production Causes Resistance to Venetoclax in Human Acute Myeloid Leukemia Via Upregulation of MCL1, BCL2A1 and BCL-XL

Introduction: Despite achievement of complete remission (CR) following chemotherapy, Acute Myelogenous Leukemia (AML) relapses in the majority of adult patients. The identification of cellular and molecular mechanisms that underlie resistance, persistence, and competitive regrowth of leukemic cells is of high interest to improve the outcome of AML patients. In this study, we have investigated the consequences of sterile leukemic inflammation on acquired novel properties and therapy resistance in human AML.

Methods: Using single cell RNA sequencing we analyzed twenty paired AML specimens procured at diagnosis and at relapse from prior CR, followed by comparative gene expression analyses of selected leukemic cell populations. In addition, we compared the single cell gene expression in leukemic monoblasts/monocytes with pooled normal monocytes of five healthy donors. We used GO analysis, pathway analysis and the database “interferome” to investigate different leukemic cell populations for enrichment of inflammatory and other signatures. We treated human non-malignant CD34+ cells with 1,000 U IFNα and IFNγ for 4h and 24h and detected gene expression changes of selected target genes via qRT-PCR. We measured the expression of IFNα, IFNb and IFNγ together with 3 retrotransposons (LINE elements) and 5 endogenous retroviruses (ERVs) in 6 immature AML (M0, M1, M2) and 8 mature AML (M4, M5a/b) via qRT-PCR. We then incubated 19 primary human AML with 1,000 U IFNα and IFNγ in 20% FBS/RPMI1640 for 4h followed by treatment with escalating doses (1 nM – 10 μM) of Venetoclax for 24 h while IFN was still present. The expression of BCL2 family members BCL2, BCL-xL, MCL1 and BCL2A1 after 4 h IFN pre-stimulation was assayed in 14 AML cases via qRT-PCR and after 24 h, 48 h and 72 h IFN treatment in 8 AML cases using immunoblotting. AML cell lines were pre-incubated with and without 500 nM of the JAK1/2 inhibitors Ruxolitinib or Baricitinib for 2 h followed by 4 h IFN pre-stimulation and measurements of Venetoclax dose responses.

Results: We have identified cellular relapse patterns and divergent maturity states of AML at diagnosis (Dx) and at relapse (Re) and found that AML comprising monoblastic/monocytic leukemias aberrantly express many inflammatory and interferon (IFN) stimulated genes. The “inflammatory response” was the most prominent gene set when comparing leukemic monoblasts with non-malignant bone marrow resident monocytes. The correlation of expression of IFN induced genes with survival uncovered extremely poor outcomes for AML patients with the highest quartile expression of the genes IL2RA, INHBA, OPTN, EPSTI1, MX1 or BST2 demonstrating a 1,000-day survival fraction of < 20% (range 4-18%). Using human non-malignant CD34+ cells treated with 1,000 U IFNα and IFNγ, we identified IL2RA, INHBA and BCL2A1 as IFN-induced target genes. We detected blast-intrinsic types I and II IFN production in chemotherapy naive AML specimens that correlated proportionally with the expression of ERVs and retrotransposons, which likely caused the IFN production. Moreover, the mean expression of multiple retroelements was substantially higher in FAB M4/M5 AML compared to M0-M2 AML. We found that one major determinant of very low expression of LINE elements and ERVs was TP53 or TET2 mutated status. Importantly, we uncovered a substantial resistance induction to Venetoclax by IFN and measured transcriptional induction of four anti-apoptotic BCL2 family members in all AML cases tested. On the protein level, we confirmed the induction of MCL1, BCL2A1 and BCL-xL by IFN in multiple AML cases suggesting a combinatorical effect of two or three anti-apoptotic BCL2 family members resulting in IFN-mediated Venetoclax resistance. Of interest, most Venetoclax sensitive AML cases lacked BCL2A1 expression. In line with these results, AML cell lines demonstrated resistance to Venetoclax following IFN stimulation, which was partially or completely reversed after JAK-inhibitor pre-treatment.

Conclusions: Our data revealed the importance of sterile inflammation for prognosis and therapy resistance in human AML. We demonstrated that IFN markedly reduces the sensitivity to Venetoclax induced AML cell death via combinatorial upregulation of MCL1, BCL2A1 and BCL-xL. Given that Venetoclax combinations are now major therapies for AML, our findings could improve our biological understanding and the therapy for adult AML.