Somatic gain-of-function mutations in members of the RAS subfamily of small guanosine triphosphatases are found in > 30% of all human cancers. We recently described a syndrome of chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity associated with a mutation in NRAS affecting hematopoietic cells, and initially we classified the disease as a variant of the autoimmune lymphoproliferative syndrome. Here, we demonstrate that somatic mutations in the related KRAS gene can also be associated with a nonmalignant syndrome of autoimmunity and breakdown of leukocyte homeostasis. The activating KRAS mutation impaired cytokine withdrawal-induced T-cell apoptosis through the suppression of the proapoptotic protein BCL-2 interacting mediator of cell death and facilitated proliferation through p27 kip1 down-regulation. These defects could be corrected in vitro by mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase 1 or phosphatidyl inositol-3 kinase inhibition. We suggest the use of the term RAS-associated autoimmune leukoproliferative disease to differentiate this disorder from autoimmune lymphoproliferative syndrome. (Blood. 2010;117(10): 2883-2886) IntroductionThe autoimmune lymphoproliferative syndrome (ALPS) is characterized by childhood onset chronic lymphadenopathy, splenomegaly, multilineage cytopenias secondary to sequestration and autoimmune destruction, and an increased risk of B-cell lymphoma. 1 Laboratory findings include polyclonal hypergammaglobulinemia and expansion of a unique population of circulating T-cell receptor ␣␤ ϩ B220 ϩ CD4 Ϫ CD8 Ϫ T (␣␤ ϩ -DNT) lymphocytes. 2,3 Most patients with ALPS harbor heterozygous autosomaldominant germline mutations in FAS, with somatic FAS mutations representing the second most common genetic cause. [4][5][6][7] Germline mutations in the genes encoding FAS ligand and caspase 10 have been identified in a small minority of patients. [8][9][10][11][12] In our cohort, approximately one-third of the patients with ALPS have an undetermined genetic basis. In addition, there is a group of genetically undetermined ALPS-like patients without ␣␤ ϩ -DNT cell elevation.We recently reported one person among these latter patients with a syndrome of lymphoproliferation, autoimmunity, and minimally increased ␣␤ ϩ -DNT cells caused by a somatic mutation in the NRAS gene, resulting in defective lymphocyte apoptosis. 13 Here, we demonstrate that somatic mutations in the homologous KRAS gene can also be associated with a syndrome consisting of autoimmune phenomena and dysregulated leukocyte homeostasis, with normal ␣␤ ϩ -DNT cells. The activating KRAS mutation, like the previously described NRAS mutation, impaired intrinsic T-cell apoptosis through the suppression of the proapoptotic protein BCL-2 interacting mediator of cell death (BIM) and facilitated cellular proliferation by repression of p27 kip1 . Methods Cells and treatmentsAll patients were studied at the National Institutes of Health (NIH) under protocols approved by the institutional review b...

Autoimmune lymphoproliferative syndrome (ALPS) is a non-malignant disease characterized by early-onset chronic lymphadenopathy, splenomegaly, multilineage cytopenias, polyclonal hypergammaglobulinemia, expansion of circulating TCRαβ+B220+CD4-CD8- T (αβ+-DNT) lymphocytes, and an increased risk of B-cell lymphoma. Most ALPS patients have germline or somatic TNFRSF6 (FAS) mutations, and a small minority of patients have germline mutations in FAS ligand and caspase 10. We recently reported that an ALPS-like phenotype can be caused by a somatic NRAS mutation (G13D) resulting in the defective lymphocyte apoptosis. Here we demonstrate that somatic mutations in the related KRAS gene can also be associated with a non-malignant syndrome of autoimmunity and breakdown of leukocyte homeostasis. The activating KRAS mutation (G13C) impaired cytokine-withdrawal induced T cell apoptosis through the suppression of the pro-apoptotic protein BIM and facilitated proliferation through p27kip1 downregulation. These defects could be corrected by MEK1 or PI3K inhibition in vitro. We suggest the use of the term RAS-associated autoimmune leukoproliferative disease (RALD) to differentiate this disorder from ALPS. In the hematopoietic system, somatic KRAS and NRAS mutations are commonly observed in aggressive tumors such as multiple myeloma or juvenile myelomonocytic leukemia (JMML). From our reports, we think patients with somatic KRAS and NRAS mutations can follow a more benign clinical course.

695 Autoimmune Lymphoproliferative Syndrome (ALPS) is an inherited disease associated with defects of lymphocyte apoptosis leading to lymphoproliferation and autoimmunity. The majority of ALPS patients have mutations in the FAS gene. MRL/lpr−/− (lpr) mice are used as a model for ALPS and are homozygous for FAS mutations, presenting with massive lymphadenopathy and splenomegaly associated with hypergammaglobulinemia, glomerulonephritis, and expansion of a rare population of TCR ab+CD4−CD8− (Double-negative, DN) T cells that are characteristic of ALPS. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS besides immunosuppressive agents like high dose steroids and rapamycin. ABT-737 is a BH3 mimetic agent that binds and inactivates anti-apoptotic proteins of the BCL-2 family such as BCL-XL and BCL-2. An oral equivalent to this drug, Navitoclax, is currently being tested in Phase 1 studies for the treatment of diverse tumors. ABT-737 is potentially of interest for the treatment of ALPS, given that it directly activates the intrinsic pathway of apoptosis, independent of the FAS pathway. We tested the BH3 mimetic ABT-737, with and without dexamethasone, in the lpr mouse model as a potential therapeutic agent for the lymphoproliferative disease. Lpr mice (N=30) were treated daily via IP injections with dexamethasone (4mg/kg) plus ABT-737 (50mg/kg) (Dex+ABT-737), dexamethasone alone (Dex), ABT-737 alone (ABT-737) or vehicle only (VEH) for 8 weeks. Vehicle treated mice developed very severe lymphoproliferation, with mean lymph node (LN) and spleen (SPL) weights of 750 mg and 465 mg, respectively. Cellularity reached LN= 85×106 cells; SPL=290×106 cells. Dex+ABT-737 treated mice had the greatest reduction in lymph node and spleen weights (final weight of 101 mg, p<0.0001; and 213 mg, p<0.005, respectively) and cellularity (LN=4×106, SPL=63×106, p<0.001), followed by ABT-737 alone (LN 453 mg, p=0.02; SPL 118 mg, p=ns; LN=20×106, p=0.001; SPL= 118×106, p=0.01), then Dex (LN 542 mg, p=ns; SPL 450mg, p=ns; LN 45×106, p=ns; SPL 241×106, p=ns). Although lymphoid cellularity was significantly lower with treatment, there was no bone marrow toxicity based on cell numbers. Additionally, there were significant reductions in DNT cells in the spleen, lymph nodes and blood with Dex+ABT-737 (p<0.005) and ABT-737 only (p<0.01) compared to vehicle. The DNT cells also appeared to be more sensitive to treatment as they were reduced to a greater extent than CD4, CD8 or B cells. ABT-737 was also able to kill patient derived EBV-transformed B cell lines in vitro, through induction of uncoupling of BAX and BAK from BCL-XL and consequent activation of the intrinsic pathway of apoptosis with release of cytochrome c and AIF. These studies provide the first evidence for the utility of this class of drugs in the treatment of a non-malignant lymphoproliferative disorder. Disclosures: No relevant conflicts of interest to declare.