Targeting SAMHD1 Promotes Anti-Tumor Immunity in Acute Myeloid Leukemia

The overall outcome of AML, which is characterized by HOXA9 overexpression (>50% cases), remains unfavorable. Success of allogeneic hematopoietic stem cell transplantation and immunotherapy highlights that T cell activation is critical for AML eradication. Notably, suppression of T cell immunity is common in the AML BM microenvironment, thus there is a pressing need to define underlying mechanisms. Recent studies reveal that the dNTP and Ara-CTP (active form of cytarabine) hydrolase SAMHD1, whose loss-of-function mutation is seen in inflammatory encephalopathy named Aicardi-Goutières syndrome, limits type-1 Interferon (IFN-I) and T cell responses by suppressing cGAS/STING signaling. Interestingly, our analysis of GEO datasets showed elevated SAMHD1 levels in specimens from AML patients relative to healthy donors, correlated with HOXA9 expression (A) and decreased T cell function.

We thus asked whether SAMHD1 levels are associated with a particular cytogenetic change or mutation upstream of HOXA9 dysregulation. Accordingly, analysis of GSE data demonstrated that AML cases with MLL rearrangement (MLL-r, t[11q23]) or NPM1 mutation (NPM1c) exhibited elevated SAMHD1 levels relative to those without either alteration (B, C). Notably, HOXA9 knockdown (KD) decreased SAMHD1 protein levels in Molm13 (MLL-r+) and OCI-AML3 (NPM1c+) cells. ChIP-Q-PCR analysis of cord blood CD34+ cells engineered with Flag-tagged HoxA9 showed significant enrichment of HoxA9 protein at the predicted SAMHD1 promoter region relative to a control site. That region also showed significant enrichment of H3K27Ac (an active transcription mark) in HoxA9-overexpressing relative to control cells. We next assessed SAMHD1 loss-of-function in MLL-AF9 (MA9) knock-in mice, first by transducing leukemia cells from MA9⁺ BM with a doxycycline (DOX)-inducible control or shSAMHD1 and then transplanting them into either WT immunocompetent or immunodeficient (Rag2^-/-) recipients to induce leukemia. Following DOX treatment, Rag2^-/- mice bearing SAMHD1-KD or control transplants exhibited comparable survival. However, WT recipients bearing SAMHD1-KD AML survived significantly longer than controls (D, E). Moreover, SAMHD1-KD-mediated leukemia ablation was reproduced in HOXA9-Meis1 transduction/transplantation AML. Furthermore, depletion of T cells, but not B cells, blocked survival advantages seen in SAMHD1-KD-transplanted animals. To determine if IFN-I responses underlie these effects, we implanted SAMHD1-KD MA9 cells into WT or Ifnar1^-/- recipients and observed significant abolition of SAMHD1-KD anti-AML effects. Moreover, SAMHD1-KD induced cGAS activation, as evidenced by elevated levels of the immune transmitter cGAMP in BM fluid from SAMHD1-KD relative to control MA9 mice. Accordingly, we observed upregulation of multiple interferon-stimulated genes (ISGs) such as ISG15 and CXCL10 downstream of cGAS/STING after SAMHD1-KD, effects blocked by cGAS deletion. To determine if immune activation requires SAMHD1 catalysis, we knocked out endogenous SAMHD1 in THP1 cells, which we then engineered cells to express doxycycline-inducible SAMHD1 variants including WT (SAMWT), catalytically-dead mutant SAMHD1 (SAMMT) or empty vector (MOCK). Comparison of ISGs expression in each group indicated that, relative to MOCK, SAMWT but not SAMMT downregulated ISGs, suggesting that blocking SAMHD1 catalysis promotes innate immunity.

To identify SAMHD1 inhibitors, we conducted a structure-based virtual screen of 8,000,000 compounds (Molport). We then assessed the top 500 candidates using cell-based phenotypic screening based on cytarabine sensitization, namely, the inhibitor should sensitize SAMWT but not SAMMT cells to Ara-C toxicity (F). We then selected the 20 best candidates to evaluate for inhibition of SAMHD1 catalysis and chose Samin27 as the most potent lead compound. Importantly, we confirmed direct Samin27 interaction with SAMHD1 protein by nuclear magnetic resonance (NMR) (G). Samin27 treatment of SAMHD1-proficient human and mouse AML lines significantly upregulated ISGs expression.

These results suggest that SAMHD1 plays an oncogenic role in AML by suppressing innate immunity. Further analysis is required to determine whether our compound can ablate AML in vivo when combined with currently available immune checkpoint inhibitors.

Salhotra:Kadmon: Other: Advisory board meeting ; BMS: Research Funding; Orca Bio: Research Funding. Marcucci:Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings.