New Anti-Cancer Drug Compounds to Treat Mutated Waldenstrom Myd88 L265P
Authors: Oliva Joan , Villanueva Larakaye , Ochiai Jun , Niihara Yutaka 
Published: November 2021

Abstract
Waldenstrom Macroglobulinemia (WM) is a non-Hodgkin lymphoma, often associated with production of monoclonal IgM in a large amount. The increased level of IgM leads to the increased level of blood viscosity, potentially causing spontaneous bleeding, headaches, vertigo and could lead to stroke and coma. WM is a rare disease, affecting around 3 cases per million per year in the USA. Different chromosomal abnormalities can be cause of WM, but the most common mutation detected in WM is the mutation L265P on the Myd88 protein, a downstream regulator of TLR4 pathway. While today there are many treatment options to manage WM, including plasmapheresis, monoclonal (rituximab) and immunomodulating drugs (halidomide), cytokines, mTOR inhibitors (everolimus), Burton tyrosine kinase (Ibrutinib), there isn't a single effective enough treatment for WM that are widely used.
In this study, MWCL-1 cells (adult WM), expressing the Myd88 L265P mutation, were used to test different drugs to monitor their effect in cell proliferation and apoptosis activities, in comparison to midostaurin (an inhibitor of FLT3), for 24 hours treatment. MWCL-1 cells were cultured in RPMI1640, in presence of 2, 10 and 25 % of fetal bovine serum (FBS). 25 % FBS was tested to be as close as possible with the level of serum present in human blood. Adipose stromal cells (ASC) were used as a reference control, in absence of serum. Compounds A (targeting TLR pathway, through IRAK-4) was used at various concentrations (0.05 to 100 µM), over 24h of treatment, in combination or not with compound B (targeting the DNA). Compound A has a high affinity for IRAK4, downstream kinase of TLR pathway. TLR pathway activation in leukemia, through Myd88 and IRAK4, is involved in the pro-inflammation response but it is also inducing proliferation and cell survival. Compound B is an inhibitor of the poly (ADP-ribose) polymerase (PARP), which are enzymes that are involved in DNA transcription, cell cycle regulation and DNA repair.
In the ASC, the caspase activity of compound A was not different from the control, but the number of cells was lower at 50 and 100 µM, indicating that the compound A was delaying the cell growth. The number of MWCL-1 cells decreased only at higher compound A concentration (50 and 100 µM), during a 24h treatment, at 2, 10 and 25 % FBS. When the cells were treated with the compound A, the level of caspase 3/7 activity was elevated only at the highest concentration (50 and 100 µM), which is consistent with the decreased number of cells, at 2, 10 and 25 % FBS. MWCL-1 were more resistant to the anti-cancer effect of the compound A at 25 % FBS, indicating that it could work on patients because 25 % FBS is close to the patient's serum level. Midostaurin, from 0.05 to 100 µM, was reducing the number of cells and increasing the caspase 3/7 activity in the cells, in a dose response manner, at 2, 10 and 25 % FBS. However, data suggest that at the highest midostaurin dose (100 µM), compound A was more efficient than midostaurin (100 µM). Compound B alone, at 10 µM, had no effect on the MWCL-1 cell number and on the caspase 3/7 activity, for the 2, 10 and 25 % FBS. However, a synergistic effect was demonstrated when compound B (10 µM) was combined with compound A at 50 and 100 µM. The combination decreased the number of MWCL-1 cells and increased the caspase activity 3/7, more so than it was compared to the single compound studies. Our data suggest that it would be possible to lower the posology of the compound A when it will be given to the patients, in combination with the compound B.
In conclusion, the data showed compound A alone or combined with the compound B decreased the level of proliferation and increased the level of apoptosis of MWCL-1 cells, cultured with 2 to 25 % of FBS. The use of compound A at high concentration to reduce the number of MWCL-1 reflect the difficulties to treat WM patient with the existing approved drugs. Further studies will be necessary to understand more the molecular mechanism affected by the compound A and/or B in the TLR-IRAK4 pathway, and to study their effect in vivo.

Disclosures
Oliva: Emmaus Lifesciences, Inc.: Current Employment. Villanueva: Emmaus Lifesciences, Inc.: Current Employment. Ochiai: Emmaus Lifesciences, Inc.: Ended employment in the past 24 months. Niihara: Emmaus Lifesciences, Inc.: Current Employment.