Corticotropin Releasing Hormone Binding Protein (CRHBP) Regulates Hematopoietic Function and Acts As a Novel AML Tumor Suppressor through a Crh-Independent Mechanism

Epigenetic deregulation is a hallmark of acute myeloid leukemia (AML) and recurring epigenetic abnormalities are present across all AML subtypes, irrespective of the underlying cytogenetic or molecular drivers. Among these, aberrant hypermethylation and silencing of CRHBP (Corticotropin Releasing Hormone Binding Protein), was seen in >90% of cases in 2 independent cohorts (n=617 AMLs). CRHBP plays a role in the hypothalamic-pituitary-adrenal (HPA) axis where it binds to Corticotropin Releasing Hormone (CRH), regulating its bioavailability. CRH, in turn, plays a role in the adjustment of neuroendocrine, autonomic, and behavioral adaptations to stress, ultimately leading to the release of cortisol. Several recent studies have shown that downregulation of CRHBP within solid tumors is associated with poor prognosis, increased metastases, and aggressiveness. Thus, we hypothesize that CRHBP plays a functional role in normal hematopoiesis and that loss of CRHBP expression may contribute to malignant transformation of hematopoietic cells.

To test this, we first confirmed that normal bone marrow CD34+ cells (NBM HSPC) in culture produce and secrete CRBHP protein by detecting the protein in the culture medium after 72h. We further validated the aberrant silencing of CRHBP by measuring it in BM plasma from NBM specimens (mean: 591.6, range: 461.7- 636.8 ng/ml; n=6) and AML patients (mean:156.7, range: 36.9-235.5 ng/ml; n=23) (p<0.0001). To confirm the epigenetic regulation of the locus, we treated 7 AML cell lines with Decitabine 500 nM x 72 h, which resulted in upregulation of CRHBP by a range of 2.0 to 120-fold change, confirming that hypermethylation is the mechanism underlying the silencing of CRHBP in AML. Next, we tested whether CRHBP plays a role in normal hematopoiesis by performing loss-of-function studies in CD34+ cells using CRISPR-Cas9 editing of the CRHBP locus compared to a non-targeting control (NTC). Efficient editing of the locus was confirmed using the T7 endonuclease I mismatch detection assay and confirming CRHBP downregulation by qRT-PCR. CRHBP-edited cells cultured in vitro (n=3) were used to test the impact of CRHBP loss on myeloid and erythroid differentiation potential. Loss of CRHBP led to a block in erythroid and myeloid differentiation, with failure to upregulate CD235a (p<0.05) and CD15 (p<0.05) by day 7. In addition, proliferation and viability analysis showed that loss of CRHBP resulted in an increase in proliferation (n = 4, p<0.01) with no impact on viability.

In the HPA axis, CRHBP binds to CRH, regulating its bioavailability in the microenvironment; however, whether CRHBP functions via CRH in HSPC and AML is not known. Notably, analysis of scRNA-seq data showed that human HSPCs do not express the canonical CRH receptors 1 and 2. We further explored whether increased levels of free CRH phenocopied the loss of CRHBP. We first quantified CRH levels by ELISA in NBM and AML BM plasma. CRH levels were 1.4-fold higher (p=0.01) in AML, with average CRH levels in AML of 1.2 ng/ml (range: 0.33 -2.07 ng/ml; n=23) and 0.86 ng/ml (range: 0.79-0.90 ng/ml; n=6) in NBM. Culturing HSPC in the presence physiological (1ng/ml) vs. AML levels (2ng/ml) of CRH did not fully recapitulate the effect that loss of CRHBP has on proliferation, resulting in mild and variable changes (n=3). Moreover, treatment with CRH failed to phenocopy the differentiation defects seen with loss of CRHBP (n=6), indicating that CRHBP may have CRH-independent roles in the BM. Next, to determine whether CRHBP may function as a soluble tumor suppressor in AML we treated 5 AML cell lines (HEL, HL60, MOLM13, OCL-AML3 and U937) with recombinant CRHBP (rCRHBP) or control every 24 h at 1X or 2X the physiological levels and compared them to normal HSPC. Notably, 4/5 cell lines showed growth inhibition by CellTiter-Glo with rCRHBP treatment while normal HSPC showed no signs of toxicity or growth difference, confirming CRHBP's selectivity against AML cells.

In summary, our findings demonstrate that CRHBP is necessary for normal HSPC function and that loss of CRHBP promotes an increase in proliferation and an impairment in differentiation. Importantly, treatment with rCRHBP induces growth inhibition in a diversity of AML cell lines, demonstrating its role as a paracrine tumor suppressor and its potential as a novel therapeutic for AML.

No relevant conflicts of interest to declare.