A novelCBL-Bflox/floxmouse model allows tissue-selective fully conditionalCBL/CBL-Bdouble-knockout: CD4-Cre mediatedCBL/CBL-Bdeletion occurs in both T-cells and hematopoietic stem cells

Abstract

CBL-family ubiquitin ligases are critical negative regulators of tyrosine kinase signaling, with a clear redundancy between CBL and CBL-B evident in the immune cell and hematopoietic stem cell studies. Since CBL and CBL-B are negative regulators of immune cell activation, elimination of their function to boost immune cell activities could be beneficial in tumor immunotherapy. However, mutations of CBL are associated with human leukemias, pointing to tumor suppressor roles of CBL proteins; hence, it is critical to assess the tumor-intrinsic roles of CBL and CBL-B in cancers. This has not been possible since the only available whole-body CBL-B knockout mice exhibit constitutive tumor rejection. We engineered a new CBL-Bflox/flox mouse, combined this with an existing CBLflox/flox mouse to generate CBLflox/flox; CBL-Bflox/flox mice, and tested the tissue-specific concurrent deletion of CBL and CBL-B using the widely-used CD4-Cre transgenic allele to produce a T-cell-specific double knockout. Altered T-cell development, constitutive peripheral T-cell activation, and a lethal multi-organ immune infiltration phenotype largely resembling the previous Lck-Cre driven floxed-CBL deletion on a CBL-B knockout background establish the usefulness of the new model for tissue-specific CBL/CBL-B deletion. Unexpectedly, CD4-Cre-induced deletion in a small fraction of hematopoietic stem cells led to expansion of certain non-T-cell lineages, suggesting caution in the use of CD4-Cre for T-cell-restricted gene deletion. The establishment of a new model of concurrent tissue-selective CBL/CBL-B deletion should allow a clear assessment of the tumor-intrinsic roles of CBL/CBL-B in non-myeloid malignancies and help test the potential for CBL/CBL-B inactivation in immunotherapy of tumors.

An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance

“…In view of reduced MG development and of MaSC depletion upon Cbl/Cbl-b DKO, we assessed whether AKT-mTOR signaling is hyperactivated in DKO MECs. As the number of cells available from the Lgr5-Cre-driven model was small, we used a fully tamoxifen-inducible Cbl and Cbl-b deletion model (Goetz et al, 2016) in which both Cbl and Cbl-b are floxed, and the ROSA26 locus CreERT and dual fluorescent reporter (mT/mG) alleles allow verifiable 4-OH-T-inducible in vitro Cbl and Cbl-b deletion. The MaSC-containing basal cells from Cbl f/f ; Cbl-b f/f ; CreERT; mT/mG or Cbl f/f ; Cbl-b f/+ ; CreERT; mT/mG mice were plated in organoid cultures and induced with 4-OH-T (Fig.…”

“…ROSA26 locus lacZ reporter [B6.129S4-Gt(ROSA)26Sor tm1Sor/J ] and EGFP reporter (CAG-CAT-EGFP) (Naramura et al, 2010) ; CreERT; mT/mG strain that was used for in vitro Cbl and Cbl-b deletion in MaSCs in certain experiments (Goetz et al, 2016). Three-week-old female NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ ) (The Jackson Laboratory) were used as recipients for mammary transplantation.…”

“…Cbl/Cbl-b DKO mammary epithelium exhibited shrinkage of the MaSC-containing basal compartment, which led us to develop a novel MaSC-specific Cbl/Cbl-b DKO model in which floxed Cbl is inducibly deleted only in Lgr5 + MaSCs. We also generated a novel mouse model in which floxed Cbl and Cbl-b can be inducibly deleted in isolated basal MECs upon tamoxifen treatment (Goetz et al, 2016). Complementary evidence from these genetic models establishes that CBL and CBL-B are redundantly required to maintain MaSCs, apparently by controlling the level of AKT-mTOR signaling.…”

CBL family E3 ubiquitin ligases control JAK2 ubiquitination and stability in hematopoietic stem cells and myeloid malignancies

“…Mice, cell lines, and primary human samples Cbl f/f ;Cbl-b f/f mice were generated as described (Goetz et al 2016) and crossed to cre ERT2 mice from Jackson Laboratories. Lnk −/− mice were generously provided by Dr. Tony Pawson and Dr. Laura Velazquez (Samuel Lunenfeld Research Institute, Toronto, Canada).…”

“…−/− and conditional Cbl) develop an aggressive CMML-like MPN due to an unexpected Cre activation in a fraction of HSCs (Naramura et al 2010). We recently generated conditional doubleknockout alleles for both Cbl and Cbl-b (Cbl f/f ;Cbl-b f/f ) (Goetz et al 2016), which show normal survival in the uninduced state. We found that pan-hematopoietic Vavcre-mediated deletion causes early lethality (day 10), preventing us from studying adult hematopoiesis .…”