Cerebrospinal Fluid Attenuates the Efficacy of Methotrexate against Acute Lymphoblastic Leukemia Cells

1. Cerebrospinal fluid (CSF) reversibly attenuates the efficacy of anti-folate drugs, including methotrexate, against leukemia cells.

2. Reduced proliferation and integrated stress response activation in leukemia cells in CSF may contribute to this resistance.

Treatment and prophylaxis of the central nervous system (CNS) is a standard component of acute lymphoblastic leukemia (ALL) therapy. However, CNS-directed therapies are a significant cause of morbidity and CNS relapse remains a cause of treatment failure. CNS-directed ALL therapies must target leukemia cells within cerebrospinal fluid (CSF), a fluid that is compositionally distinct from plasma and has been shown to impact leukemia biology. Herein, we demonstrate that human CSF attenuates the potency and efficacy of anti-folate drugs including methotrexate, the primary CNS-directed chemotherapeutic for over six decades. Importantly, this effect of CSF on leukemia methotrexate sensitivity was reversible. Additional mechanistic studies support that diminished proliferation and activation of the integrated stress response (ISR) in leukemia cells in CSF may contribute to this resistance. Our findings suggest potential strategies to enhance methotrexate efficacy in CNS-directed ALL therapy and highlight the need to critically reassess even established standards of care.