Oncogene addiction refers to a cancer cell’s dependence on the continued activity of a single oncogene for survival. This concept has been validated by the success of tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML), a clonal myeloproliferative disorder characterized by the presence of the BCR-ABL1 oncoprotein. BCR-ABL1 is a tyrosine kinase that constitutively activates RAS, which initiates multiple downstream signaling pathways that regulate cell proliferation, differentiation, and apoptosis. Because aberrant regulation of these cellular processes results in disease progression and resistance to therapy, a better understanding of how BCR-ABL1 activates RAS may uncover new therapeutic strategies. Previous studies in our lab have demonstrated that BCR-ABL1 mediated oncogene addiction is facilitated by persistent levels of MEK-dependent negative feedback, which oppose BCR-ABL1-dependent RAS activation. Inhibition of MEK in BCR-ABL1 expressing cells increases RAS-GTP levels, thereby providing a tractable, disease-relevant model to address the question of how BCR-ABL1 activates RAS. Here we identify a biologically and therapeutically important novel mechanism whereby BCR-ABL1 activates the critical downstream effector RAS in CML cells. We find that BCR-ABL1 activates RAS in part through the PLCG1/DAG/RasGRP4 signaling axis. Loss of PLCG1 decreases RAS nucleotide exchange factor activity, decreases cell proliferation, and increases sensitivity to BCR-ABL1 tyrosine kinase inhibitors (TKIs). Collectively, these studies suggest that PLCG inhibitors may re-sensitize some patients to BCR-ABL1 TKIs, and may lead to more effective cytotoxicity when combined with BCR-ABL1 TKIs.

Another mechanistic insight of BCR-ABL1 mediated oncogene addiction our lab has reported is that the negative feedback established by BCR-ABL1 persists for an extended period of time following the initiation of TKI treatment, during which CML cells commit to apoptosis. This is in contrast to what has been observed in BRAF-V600E driven malignancies, where MEK-dependent negative feedback rapidly attenuates following BRAF inhibitor treatment. This observation has raised the question of whether negative feedback can be harnessed to affect targeted therapy response in pathologically activated kinases. We sought to identify the critical molecular entities responsible for the prolonged negative feedback in patient-derived CML cell lines. We identified some candidate mediators of prolonged negative feedback in BCR-ABL1-expressing cells and validated that they were able to affect BCR-ABL1 TKI sensitivity. Future studies will need to be performed to determine the extent to which these candidate genes modulate the duration of negative feedback.

Lymphocytes express two Ras guanine exchange factors (RasGEFs), Sos and RasGRP1, which can act in concert to activate the Ras-MEK-ERK pathway downstream of antigen receptor signaling. RasGRP1 is capable of priming Sos and is required for optimal Sos activation. Sos is incapable of compensating for loss of RasGRP1 in lymphocytes as RasGRP1-deficient mice fail to develop single positive thymocytes. Conversely, overexpression of RasGRP1 in the thymus leads to the development of thymomas. Since RasGRP1 exists in this critical position between immunodeficiency and oncogenesis, it is important to understand the mechanisms underlying the regulation of RasGRP1 protein levels and activity.

My studies suggest that posttranslational modifications of RasGRP1 exist to limit RasGRP1 activity after antigen receptor stimulation. RasGRP1 appears to be monoubiquitinated and is phosphorylated in the basal state as well as upon TCR stimulation. Additionally, a hypomorphic allele of RasGRP1 (RasGRP1Anaef) leads to relatively normal thymocyte development in the context of a wildtype TCR repertoire, but a modest impairment in positive selection is revealed when a transgenic TCR is used. Surprisingly, mice expressing this allele have appear to have phenotypically activated T cells, produce anti-nuclear antibodies and have hypercellular spleens by the age of 12 weeks. It appears that RasGRP1 may play a role in negatively regulating TCR signaling that was previously unrecognized. Thus, RasGRP1 must be properly regulated to prevent autoimmunity as well. Further work examining RasGRP1 protein function and regulation is required to understand the possibility and implications pharmacological targeting of this critical RasGEF in the disease state.

To maintain immunological tolerance and prevent autoimmunity, naïve CD4+ T cells need to be able to discriminate between self and nonself. In addition, naïve T cells need to remain quiescent and prevent spontaneous activation or differentiation, yet they also must be primed to efficiently respond when they do encounter foreign antigen. These features suggest that there must be active mechanisms to regulate the naïve state of CD4+ T cells. We have uncovered that tonic (basal) signaling pathways in peripheral CD4+ T cells dynamically maintain this primed-yet-quiescent state in three manners: transcriptional, translational, and metabolic programs.

We identified a tonic pathway involving the adapter LAT that dynamically controls a transcriptional program in naïve CD4+ T cells. LAT perturbation in T cells leads to a spontaneous Th2-biased lymphoproliferative disease. We found that LAT is critical for tonic regulation of the repressor HDAC7. Tonic signals lead to phosphorylation and cytoplasmic localization of HDAC7. Without basal LAT function, HDAC7 is dephosphorylated and resides in the nucleus, where it represses target genes such as Nur77 and Irf4. Appropriate expression of these genes is important for preventing spontaneous proliferation and differentiation of CD4+ T cells.

Second, we identified a connection between tonic Rasgrp1-mTOR signals and translational programs. CD4+ T cells with a point mutation in the Ras activator Rasgrp1, called Rasgrp1Anaef, exhibit increased basal mTOR signaling and as a result the mice develop autoimmune features. We found that Rasgrp1Anaef CD4+ T cells have an autoreactive TCR repertoire and show increased differentiation to Tfh- and Th2, with concomitant increase in Gata3 protein levels that we hypothesize is caused by increased translation. We are currently investigating what global basal translation programs are regulated by tonic Rasgrp1-mTOR signals in naïve T cells.

Additionally, we are following up on findings that tonic mTOR signals control the metabolic state of naïve T cells and allow for efficient switching of metabolic programs upon T cell activation. Overall, our results provide novel insights into the role of tonic signaling pathways in controlling naïve CD4+ T cells and how point mutations in signaling proteins can disrupt these tonic pathways, leading to perturbed immune function.

Mammary stem cells (MaSC) are a heterogeneous population that give rise to progenitor cells subsequently developing into differentiated cells. The exact mammary epithelial cell (MEC) hierarchy and particularly the signals that govern this hierarchy are largely unknown. Several studies have implied a role for EGFR signals in the mammary epithelium but the exact role that of EGFR (Epidermal Growth Factor Receptor) has remained rather elusive. Insights from a 3-D ex vivo model derived from human breast tissue and studies using a hypomorphic EGFR allele have put forth a model that the strength of EGFR signals may impact cell fate decisions in the mammary gland. We obtained recent evidence indicating that the Ras guanine nucleotide exchange factor (RasGEF) Rasgrp1 can dampen EGFR signals in epithelial cells.

We demonstrate that RasGEF Rasgrp1 is expressed in mammary epithelium and use cleared fat pad approaches to reveal a previously unrecognized functional role for RasGRP1 in the mammary gland. These approaches involved the transplantation of mammary epithelial cells (MECs) from Rasgrp1-deficient animals and from mice with a single point mutation in Rasgrp1, the Rasgrp1Anaef model. Biochemically, loss of Rasgrp1 as well as the Rasgrp1Anaef point mutation resulted in increased EGF-induced Ras-effector kinase signaling. MECs showed increase activation of the ERK-, Akt-, and mTORC1-S6- kinase pathways upon EGF stimulation when the MECs were isolated from Rasgrp1-/- or the Rasgrp1Anaef females. Using a model cell line, Eph4, we demonstrated that Rasgrp1’s suppressive action depends on its catalytic activity.

Functionally, Rasgrp1 perturbation led to shortened mammary ductal trees in mammary whole mounts, increases in numbers of proliferative terminal end buds (TEBs), and sustained proliferation of mammary ductal cells (with high phospho-Akt) in areas where proliferation has normally halted. Organoid assays to reveal that Rasgrp1 perturbation led to a gain-of- function EGF-driven phenotype. With WT MECs, EGF drove the formation of spheres in organoid assays. By contrast, EGF induced robust branching of Rasgrp1-/- and Rasgrp1Anaef MECs that could be prevented when EGFR inhibitors are added. Thus, Rasgrp1 critically controls the mammary morphogenetic program as indicated by elevated branching morphogenesis upon EGF-EGFR signaling in the absence of Rasgrp1 function.

MEC colony assays using Rasgrp1-/- and Rasgrp1Anaef cells revealed that progenitors form more and larger colonies than WT counterparts and with increased proliferation (Ki67) in response to EGF and strong staining for pAkt, all of which were reduced with EGFR inhibitors in the assays. Lastly, capitalizing on FACS profiling of mammary epithelial cells we established that Rasgrp1 perturbation led to alterations in cell fate of stem- and progenitor-cells in the mammary gland. Rasgrp1-/- and Rasgrp1Anaef mammary glands revealed decreased total cellularity of CD31-/CD45-/Ter119- lineage-negative cells but increased luminal CD49fmedium/EpCAMhigh and basal CD49fhigh/EpCAMmedium cell numbers that encompasses the MaSCs.

In sum, we provide mechanistic insights on the biochemical signals that impact MACSs and progenitor cells. We show that Rasgrp1 is required to balance MEC EGFR signals and regulate mammary stem and progenitor cell fate to dampen proliferation so that cells can exit mitosis and maturate and differentiate.