Synergistic Effect of Tamoxifen and Ruxolitinib Combination in Reducing Disease Burden in Myeloproliferative Neoplasms
Authors: Pereira-Martins Diego A , Alves-Silva Antonio Bruno , Albuquerque De Melo Manuela , Fonseca Natasha Peixoto , Weinhäuser Isabel , Perez Fabio Augusto Bueno , Bellomi Artur Bernardo Ribeiro , Figueiredo-Pontes Lorena Lobo , Machado-Neto João Agostinho , Quek Lynn , Chatzikyriakou Prodromos , Huls Gerwin A. , Ammatuna Emanuele , Schuringa Jan Jacob , Traina Fabiola 
Published: December 2024

Myeloproliferative neoplasms (MPN) are often associated with mutations in JAK2, CALR, and MPL, leading to the constitutive activation of the JAK2/STAT signaling pathway. The FDA-approved JAK1/2 inhibitor Ruxolitinib (Ruxo) is used to treat specific MPN patients, offering good tolerability and a reduction in constitutional symptoms. However, Ruxo effect on reducing tumor size is limited. The TAMARIM trial (Fang et al., Nat Comm 2023) showed that tamoxifen monotherapy (Tam) reduced the mutant allele burden in 13% of the patients. Here, we explored whether combining Ruxo and Tam could provide superior disease control in preclinical MPN models, using ex vivo treated primary MPN samples and in vivo models. In vitro, the combination therapy of Ruxo (1-10 µM) and Tam (0.3-3 µM) over 72 hours exhibited strong synergistic effects (ZIP score 24.4) in the HEL cell line, an MPN model with the JAK2V617F mutation. Treatment with either Ruxo or Tam alone significantly decreased the mitochondrial membrane potential (MMP) and both basal and maximum oxygen consumption rates in HEL cells. However, these effects were significantly enhanced with the combination of Ruxo and Tam. These results suggest modulation of the mitochondrial metabolism as a common mechanism of both drugs in MPN cells. For primary MPN samples, we performed ex vivo drug screening on 20 bone marrow (BM) samples from MPN patients (median age [range]: 68 [46-82] years; 53% males). The samples included diagnoses of essential thrombocythemia (13%) and myelofibrosis (MF, 87%). Ruxo monotherapy (0.3-1 µM) did not significantly induce apoptosis. In contrast, Tam monotherapy or in combination with Ruxo significantly induce apoptosis and downregulated MMP in MPN blasts (CD34+ cells). Functional respiration analysis confirmed a decrease in mitochondrial metabolism in both Tam monotherapy and the Ruxo+Tam combination in ex vivo treated MPN samples. A subset of cells showed decreased sensitivity to Tam, particularly in patients with secondary MF. Flow cytometry analysis identified a population of MPN blasts expressing the fatty acid transporter CD36, associated with resistance to Tam monotherapy. This resistance could be overcome by the combination with Ruxo. To explore the potential effects of the combination therapy of Ruxo+Tam in vivo, we transplanted 5x10e6 BM mononuclear cells from a JAK2V617F murine model (CD45.2) into lethally irradiated B6 PepBoy mice (CD45.1). Four weeks post-transplant, peripheral blood (PB) chimerism and hematological counts were assessed, and mice were divided into 4 groups for treatment: vehicle, Tam (75 mg/kg, intraperitoneal injection), Ruxo (120 mg/kg, oral gavage twice daily), and the combination Ruxo+Tam. Mice were treated daily for 8 weeks, with good tolerability and no significant weight loss. PB samples were collected bi-weekly to monitor blood chimerism, immune subset frequencies, and hematological counts. PB chimerism analysis showed a significant reduction in CD45.2 cells (JAK2V617F mut) beginning at week 6 in the Ruxo+Tam group, with no significant changes observed in the Ruxo or Tam monotherapy groups compared to vehicle control. Analysis of immune subsets in the Ruxo+Tam group revealed a reduction in the myeloid cell fraction within the CD45.2 population, which was linked to decreased MMP, suggesting modulation of mitochondrial metabolism in these cells. Additionally, the combination therapy resulted in a significant decrease in CD36+ myeloid cells within the CD45.2 population by week 2. Hematological analysis indicated a strong reduction in leukocyte, platelets, and hematocrit levels starting from week 2 in the Ruxo+Tam group, indicating superior disease control with the combination therapy. In summary, our data support the rationale for using the combination therapy of Ruxo+Tam in treating MPN. We demonstrate that both drugs likely act by modulating mitochondrial metabolism, an effect that is enhanced when used in combination, thereby overcoming resistance seen with monotherapies. Resistance to Tam was observed in patients with high CD36 levels, which could be countered by combining it with Ruxo. Additionally, since Tam is a relatively inexpensive drug, its incorporation into clinical practice could be particularly feasible and beneficial in low- and middle-income countries. These findings support further clinical studies to explore the efficacy of this combination therapy for MPN patients.