Program Director/Principal Investigator (Lachman, Herbert M)
Abstract
Mutations in PPM1D (Protein Phosphatase Magnesium-Dependent 1 Delta), which codes for a member of the
protein serine-threonine phosphatase family, have recently been found in individuals with Jansen-De Vries
Syndrome (JdVS), a condition characterized by intellectual disability (ID), severe anxiety, oppositional
behavior, attention problems, high pain threshold, restricted eating, and gastrointestinal problems. So far,
several dozen children with JdVS have been reported in the literature. Remarkably, each has a de novo
germline nonsense or frameshift mutation in exon 5 or 6 that predicts the formation of a protein truncated at the
C-terminal end. The phosphatase domain is spared. A key question is whether the truncating mutations result
in gain- or loss- of PPM1D function in neurons and other brain cells. PPM1D is also a tumor suppressor gene
that is over-expressed in a variety of cancer subtypes, contributing to malignant transformation through its
action as a negative regulator of the p53-mediated DNA repair pathway. In cancers, somatic, truncating
mutations in exons 5 and 6 like those found in JdVS, but somatic in origin, result in gain-of-function (GOF)
effects because PPM1D degradation is reduced. Yet, loss-of-function (LOF) variants in the catalytic domain
have also been found in some cancers, suggesting that the effects of PPM1D on cellular function could be
context-dependent. Whether the germline truncating mutations in children with JdVS cause GOF or LOF is a
critical question to address because PPM1D inhibitors being developed by cancer researchers could
potentially have therapeutic value in JdVS if the former is correct. On the other hand, if they cause LOF, drugs
targeting over-phosphorylated downstream targets caused by reduced PPM1D phosphatase activity, would be
a more feasible approach. To address these fundamental questions, we have established an induced
pluripotent stem cell (iPSC) model for JdVS using patient-specific and CRISPR-engineered lines, each
containing exon 5 or exon 6 truncating mutations. Preliminary RNA-seq and proteomics experiments on
glutamatergic neurons derived from iPSCs identified a few functional signatures, including deficits in neurite
outgrowth, altered chromatin architecture, and calmodulin kinase 2 (CAMK2) phosphorylation that suggest a
GOF effect. However, molecular studies need to be repeated and expanded in additional lines to firmly
establish these as legitimate phenotypes, and to identify novel makers that can be used to both test the GOF
hypothesis and identify downstream targets for therapeutic intervention. This will be accomplished by a multi-
OMICs approach on glutamatergic neurons that includes proteomics, phosphoproteomics, RNA-seq, and
ATAC-seq. Considering the uniqueness of the PPM1D mutations in the development of JdVS, we also
hypothesize that truncated PPM1D proteins have powerful effects on cellular function that could make them
resistant to typical PPM1D inhibitors. To test these hypotheses, we will knockout either the mutant allele or wild
type PPM1D allele by introducing a null mutation using CRISPR-Cas9 editing, after which, rescue of
dysregulated cellular and molecular phenotypes will be assessed. The ability of small molecule PPM1D
inhibitors to rescue these phenotypes will also be evaluated. The experiments will show whether JdVS
associated PPM1D variants have a GOF effect on glutamatergic neurons, and whether generalized reduction
in PPM1D phosphatase activity or targeted inhibition of the truncated protein will be suitable targets for
therapeutic intervention.
