CUX1 haploinsufficiency antagonizes apoptosis to sustain AML

Monoallelic loss of CUX1 confers poor prognosis, but its role in acute myeloid leukemia (AML) development remains obscure. Here, we show that CUX1 deletion induces CFLAR-mediated apoptosis inhibition crucial for AML survival and establish CFLAR as a therapeutic target in this high-risk AML subtype.

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In our previous work, we performed a comprehensive analysis of DNA sequences of 7,651 tumors from 28 cancer types to identify the transcription factor cut-like homeobox 1 (CUX1) as a novel haploinsufficient tumor suppressor gene (TSG)1, contrary to its assigned oncogenic roles that were known at that time2,3. In our study, we searched for genes showing an enrichment in loss-of-function mutations to provide us with a landscape of putative TSGs, including those occurring at a low frequency such as CUX1, which would have otherwise escaped detection in smaller scale sequencing studies. Specifically, we determined that truncating mutations in one allele of the CUX1 gene were recurrent in myeloid malignancies such as AML1. Notably, CUX1 lies in a region of chromosome 7q, which is recurrently lost through complete or partial monoallelic deletions [-7/del(7q)] in several types of myeloid malignancies, implying that CUX1 is an important chromosome 7q target gene in these diseases4.

Following the identification of CUX1 as a haploinsufficient TSG, we were faced with two important questions that we sought to address in the present study: firstly, was it possible to identify vulnerabilities specific to CUX1-deficient AML that could be exploited for AML therapy? Since both -7/del(7q) lesions and CUX1 haploinsufficiency confer a poor prognosis in AML and other myeloid malignancies1,5, there is an unmet clinical need for targeted therapies in these high-risk cases. Secondly, how did CUX1 haploinsufficiency contribute to leukemia development and did it require cooperating mutations? While the role of biallelic TSG inactivation in tumorigenesis has been widely acknowledged since Knudson`s two-hit hypothesis from 19716, the function of TSG haploinsufficiency in cancer remains less well described, partly due to the lack of genetic mouse models, which can recapitulate haploinsufficiency accurately in vivo7,8.

To address the first question, we performed CRISPR/Cas9 drop-out screens in isogenic wild-type and CUX1-knockout AML cell lines, which identified the CASP8 and FADD-like apoptosis regulator (CFLAR) gene as a preferential vulnerability in CUX1-deficient cells (Figure 1). The encoded anti-apoptotic protein CFLAR (also known as cFLIP) inhibits the extrinsic apoptosis pathway by competing with pro-caspase-8 for binding to death receptors, thereby preventing caspase activation9. Mechanistically, we could show that the full-length isoform of CUX1 acts as a direct transcriptional repressor at the CFLAR promoter, providing insight into how CUX1 deficiency upregulates CFLAR expression to safeguard against apoptosis.

Figure 1. CRISPR/Cas9 screens identify CFLAR as a selective vulnerability in CUX1-deficient AML. Scheme of CRISPR/Cas9 dropout screens (left) and distribution of residual scores as a measure of gene essentiality in CUX1-deficient U937 cells (right).

To address the second question, we next sought to generate a conditional Cux1-haploinsufficient mouse model. However, we quickly realized that heterozygous Cux1 deletion alone was insufficient to promote AML development, suggesting the requirement for additional genetic mutations. Therefore, we searched for mutations cooccurring with -7/del(7q) lesions in human AML and chose FLT3-internal tandem duplication (FLT3ITD) mutations, which are found in ~10% of -7/del(7q) cases of AML10, for further investigation. Combining Cux1 haploinsufficiency with Flt3ITD mutation resulted in a lethal leukemia in all double-mutant mice (Cux1+/-;Flt3ITD) with a median survival of 28 weeks. Interestingly, while moribund Cux1+/-;Flt3ITD mice culled at an earlier age were diagnosed with a disease similar to human chronic myelomonocytic leukemia (CMML), older animals exhibited features of AML (Figure 2). In line with our previous CRISPR/Cas9 screen in human cells, genetic depletion of Cflar preferentially diminished the survival of Cux1-deficient cells in vitro and in vivo, supporting our hypothesis that Cflar is a selective vulnerability in the context of Cux1 deficiency. Combined, these results indicate that CUX1 haploinsufficiency cooperates with FLT3ITD mutation to promote AML, which is associated with an induction in CFLAR-mediated anti-apoptosis pathways.

Figure 2. Mouse model of CMML and AML. Survival curves of control (Con), Cux1+/-, Flt3ITD and double mutant Cux1+/-;Flt3ITD mice.

To investigate whether targeting of CFLAR can be translated into AML therapy, we wanted to assess the effects of pharmacological CFLAR inhibition on CUX1-deficient cells. However, since direct CFLAR inhibitors are not routinely available, we tested the impact of the second mitochondrial-derived activator of caspases (SMAC)-mimetic drug birinapant ( on Cux1-haploinsufficient cell survival as an alternative therapeutic approach (Figure 3). Indeed, murine hematopoietic cells from Cux1-haploinsufficient mice as well as primary human AML samples harboring -7/del(7q) lesions displayed higher sensitivity to birinapant compared with their wild-type and normal karyotype counterparts, respectively.

Figure 3. Mechanism of CUX1/CFLAR-mediated apoptosis regulation. A. Under physiologic conditions, the tumor suppressor and transcription factor CUX1 represses the expression of CFLAR. B. In CUX1-deficient cells, transcriptional repression of CFLAR is alleviated leading to its expression and inhibition of the extrinsic apoptosis pathway. DD, death domain; FADD, FAS-associated death domain; DISC, death-inducing signaling complex; XIAP, X-linked inhibitor of apoptosis. (Modified from Cassier et al.)11

Finally, considering that birinapant in combination with azacitidine for the treatment of genetically unselected patients with higher-risk myelodysplastic syndrome or CMML failed to improve response rates compared with azacitidine alone12, our findings suggest that clinical studies evaluating SMAC-mimetics such as birinapant could benefit from a more stringent stratification of patients. Therefore, we encourage not only the development of direct CFLAR inhibitors but also the further clinical evaluation of SMAC-mimetics specifically in CUX1-haploinsufficient and -7/del(7q) myeloid malignancies.



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Saskia Rudat

Postdoctoral Research Fellow, Wellcome Sanger Institute