Functional Analysis of MYC Deregulation By Diverse Genetic Mechanisms during Hematopoiesis

Alterations of the c-MYC (MYC) locus, mainly in the form of translocations or amplifications, are common in hematologic malignancies and result in deregulated MYC expression, disrupting cellular proliferation and differentiation and promoting leukemogenesis. While amplified MYC loci typically maintain all native cis-regulatory elements, translocated loci are instead driven by heterologous regulatory regions and are therefore expected to be unresponsive to external signals. However, no studies have directly compared the effects of different modes of MYC deregulation in cellular phenotypes and functions.

Consistent with the idea that different modes of MYC deregulation may respond differentially to external mitogenic signals, we found that treatment with DMSO resulted in rapid downregulation of MYC protein expression in hematopoietic cell lines with amplified MYC loci (HL-60, BJAB), but failed to do so in cell lines harboring a t(8;14) MYC translocation (Ramos, LY8) despite the induction of cell cycle arrest in all lines. To precisely model these two genetic mechanisms of MYC deregulation during hematopoiesis and study their effects in phenotypes relevant to oncogenesis (differentiation and proliferation), we used genetic engineering of human embryonic stem cells (hESCs). First, MYC deregulation upon translocation was modeled using a doxycycline (DOX)-inducible lentiviral construct to ectopically express MYC with GFP (MYC-DoxOE). Transduced single cell clones of the H1 hESC line were selected to ensure uniform MYC expression. Second, we created a conditional MYC gene amplification model in H1 hESCs by using CRISPR/Cas9 to insert a floxed cassette consisting of three tandem MYC cDNA copies and GFP in the MYC locus (MYC-Amp). Correctly edited clones were identified via PCR and Southern Blot, and one clone with bi-allelic targeting (yielding 8 inducible MYC copies) was selected. All clones were confirmed to be karyotypically normal.

Upon in vitro hematopoietic differentiation of unmodified H1 hESCs, we identified a peak of MYC expression (day 12) coinciding with the emergence of CD34⁺/CD45⁺ hematopoietic progenitor cells (HPCs). MYC expression subsequently declined as cells matured further down the myeloid lineage and lost CD34 expression (days 14-20). To test the effects of MYC deregulation in the two models we induced MYC expression by doxycycline (MYC-DoxOE) or cell-permeant TAT-Cre recombinase (MYC-Amp) on day 10 of hematopoietic differentiation. Induction of MYC expression above normal levels was found to be comparable between the two models during peak MYC expression (day 12). However, while ectopically expressed MYC in the MYC-DoxOE model maintained high levels throughout the culture period, amplified MYC in the MYC-Amp model - which is subject to regulation by the native MYC locus cis regulatory elements - was downregulated after day 12, in a pattern similar to that observed in uninduced H1 cells.

MYC induction in the MYC-DoxOE model caused a block in differentiation with accumulation of CD34⁺ HPCs and reduction of more mature CD34^-/CD45⁺ hematopoietic cells. Wright-Giemsa staining corroborated these results showing a predominance of morphologically immature myeloid cells. Strikingly, MYC-DoxOE HPCs could be maintained in culture for over 90 days, while H1-derived HPCs without doxycycline treatment completely arrested their proliferation after approximately 28 days of culture. This phenotype is dramatically distinct from the finite growth potential of HPCs derived from normal human pluripotent stem cells, documented in multiple hESC and induced pluripotent stem cell (iPSC) lines, and reminiscent of the extended proliferation we previously reported in HPCs derived from iPSCs generated from patients with acute myeloid leukemia (AML) (Kotini et al. Cell Stem Cell 2017). Induction of MYC in the MYC-Amp model had similar effects in differentiation and proliferation, albeit much less pronounced, extending the growth of HPCs to approximately 50 days. This milder phenotypic perturbation is likely due to the downregulation of amplified MYC, as opposed to the continuous high expression of the ectopically expressed MYC in the MYC-DoxOE model.

In summary, we have generated a novel model that offers for the first time the opportunity to study the effects of MYC deregulation by different genetic mechanisms in human hematopoiesis in a dynamic fashion.