Proteins are molecular tools that are required for general cell maintenance and function. Our bodies rely on a process called ubiquitination to control how proteins are used and removed. When this process goes wrong, it can lead to the buildup of proteins, which can result in diseases including cancers and neurodegenerative disorders. Ubiquitination is regulated by enzymes called E1 ubiquitin activating enzymes (E1s), which initiate an enzyme cascade involving other ubiquitin enzymes (E2s and E3s) to maintain proper protein levels. This process, known as protein homeostasis, is crucial for healthy blood cell formation. Blood stem cells and their offspring require fine-tuned protein levels to maintain cellular function to allow for healthy white and red blood cell formation. Without this control, blood cells, such as immune cells, cannot properly function leading to poor outcomes in these patients. Additionally, failure to maintain protein homeostasis is directly linked to some blood cancers. Cancer treatments targeting this protein maintenance system have been developed, but they are not effective for everyone and can have serious side effects. While scientists have studied how some E2 and E3 enzymes are involved in blood cancers, the role of E1 enzymes in blood disease is still unclear.

 One disease that may help us understand the role of E1s in disease is VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, a condition that primarily affects 1 in 4000 men over the age of 50. It is characterized by severe inflammation, immune cell problems, and often leads to bone marrow failure. Because of this, long term survival for these patients is quite poor. Many people with VEXAS also develop a type of blood cancer called myelodysplastic syndrome (MDS) further complicating their clinical outcomes. VEXAS is caused by mutations in a gene called UBA1, which is one of two E1 ubiquitin enzymes expressed in humans. These mutations alter ubiquitination in blood cells resulting in aberrant protein expression and cause widespread blood cell dysfunction. Currently, treatment options for VEXAS are limited and mostly focus on reducing the effects of high inflammation through steroid therapies. Some drugs used for MDS treatments can be used for a subset of eligible patients, but there is a clear need for better therapies that directly target mutant UBA1 cells.

 Here, we describe the development of a new human cell line model of VEXAS using a human leukemia cell line (THP-1). We introduced a common VEXAS causing UBA1 mutation into these cells and found that mutant UBA1 cells mimicked key features of the disease, including vacuoles, abnormal protein buildup, and inflammation. This model led to key observations of the functional role of UBA1 mutations in the regulation of protein homeostasis and inflammation. Interestingly, we find that while the total level of ubiquitination is similar between normal and mutant cells, the extent to which certain proteins are regulated by ubiquitination in mutant cells is altered. Thus, the symptoms of VEXAS syndrome may not simply be caused by the general accumulation of proteins but rather by a subset of proteins that are regulated differently in mutant cells. We also find that while inflammation is a key feature of VEXAS, the UBA1 mutant cells actually reduce the activity of some immune-related genes. This might be a survival strategy, helping the mutant cells live through constant inflammation while healthy cells die off. This could explain how mutant UBA1 cells take over the bone marrow, leading to bone marrow failure in these patients.

To explore new therapeutic avenues in VEXAS syndrome, we tested whether these mutant cells were more sensitive to inhibition of the two E1 ubiquitin enzymes UBA1 and UBA6. We utilized two strategies, short hairpin RNAs (shRNA), molecules that can effectively reduce the ability for a gene to be expressed, and small molecules, drugs that can reduce the activity levels of target proteins. We find that shRNA knockdown of UBA1 resulted in a strong reduction in cellular function for both normal and mutant cells, suggesting that therapies targeting UBA1 directly may be difficult to develop. We next looked into the effects of the reduction of UBA6 in normal and mutant cells. We find that mutant UBA1 cells are sensitized to loss of UBA6 compared to normal cell controls. To further test this, we utilized two small molecules, TAK-243 and phytic acid, which inhibit UBA6 protein activity. We find that phytic acid, a compound which naturally occurs in grains and legumes, was able to suppress mutant UBA1 cell growth in both cell lines and in mouse studies. Taken together, we have utilized a new cell model of VEXAS syndrome to identify UBA6 inhibition as a new therapeutic avenue to explore.

 Key Findings:

• Development of a new human cell line model of VEXAS syndrome recapitulates key features of the disease
• Mutant UBA1 results in alterations to ubiquitinated proteins and inflammation
• Cells with UBA1 mutations are more sensitive to UBA6 inhibition

Natural Compound Shows Promise:

• Phytic acid (IP6), found in grains and legumes, was tested as a natural UBA6 inhibitor
• IP6 selectively killed UBA1-mutant cells while sparing healthy ones.
• In mice, IP6 extended survival in mice injected with UBA1-mutant cells and was well tolerated

Why This Matters:

• This creates a unique vulnerability that can be targeted with drugs like IP6 or TAK-243.
• These findings open the door to new, more precise treatments for VEXAS syndrome.