Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.

The Raf/MEK/ERK cascade is a therapeutic target in human cancers with deregulated Ras signaling, which includes tumours that have inactivated the Nf1 tumour suppressor1. Nf1 encodes neurofibromin, a GTPase activating protein that terminates Ras signalling by stimulating hydrolysis of Ras•GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed due to outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4, and Mapk14, which encodes p38α. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

Oncogenic K-Ras proteins, such as K-RasG12D, accumulate in the active, guanosine triphosphate (GTP)–bound conformation and stimulate signaling through effector kinases. The presence of the K-RasG12D oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf–mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) and phosphoinositide-3 kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. Here, we show that phospholipase C γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population highly enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-RasG12D remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

Trabecular bone mineral content was assessed by quantitative computed tomography in eleven young adults with phenylketonuria who had been treated from early childhood with a diet restricted in natural protein and supplemented with amino acids, minerals and vitamins. There was a significant reduction in the bone mineral content of patients compared with the normal population. Prospective studies are indicated in younger patients to ensure optimum bone mineralisation is achieved by adulthood.