IL-38 blockade induces anti-tumor immunity by abrogating tumor-mediated suppression of early immune activation.

Immune checkpoint inhibitors that overcome T cell suppressive mechanisms in tumors have revolutionized the treatment of cancer but are only efficacious in a small subset of patients. Targeting suppressive mechanisms acting on innate immune cells could significantly improve the incidence of clinical response by facilitating a multi-lineage response against the tumor involving both adaptive and innate immune systems. Here, we show that intra-tumoral interleukin (IL)-38 expression is a feature of a large frequency of head and neck, lung and cervical squamous cancers and correlates with reduced immune cell numbers. We generated IMM20324, an antibody that binds human and mouse IL-38 proteins and inhibits the binding of IL-38 to its putative receptors, interleukin 1 receptor accessory protein-like 1 (IL1RAPL) and IL-36R. In vivo, IMM20324 demonstrated a good safety profile, delayed tumor growth in a subset of mice in an EMT6 syngeneic model of breast cancer, and significantly inhibited tumor expansion in a B16.F10 melanoma model. Notably, IMM20324 treatment resulted in the prevention of tumor growth following re-implantation of tumor cells, indicating the induction of immunological memory. Furthermore, exposure of IMM20324 correlated with decreased tumor volume and increased levels of intra-tumoral chemokines. Together, our data suggest that IL-38 is expressed in a high frequency of cancer patients and allows tumor cells to suppress anti-tumor immunity. Blockade of IL-38 activity using IMM20324 can re-activate immunostimulatory mechanisms in the tumor microenvironment leading to immune infiltration, the generation of tumor-specific memory and abrogation of tumor growth.

Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice.

Human leukemic stem cells, like other cancer stem cells, are hypothesized to be rare, capable of incomplete differentiation, and restricted to a phenotype associated with early hematopoietic progenitors or stem cells. However, recent work in other types of tumors has challenged the cancer stem cell model. Using a robust model of xenotransplantation based on NOD/SCID/IL2Rγc-deficient mice, we confirmed that human leukemic stem cells, functionally defined by us as SCID leukemia-initiating cells (SL-ICs), are rare in acute myelogenous leukemia (AML). In contrast to previous results, SL-ICs were found among cells expressing lineage markers (i.e., among Lin+ cells), CD38, or CD45RA, all markers associated with normal committed progenitors. Remarkably, each engrafting fraction consistently recapitulated the original phenotypic diversity of the primary AML specimen and contained self-renewing leukemic stem cells, as demonstrated by secondary transplants. While SL-ICs were enriched in the Lin-CD38- fraction compared with the other fractions analyzed, SL-ICs in this fraction represented only one-third of all SL-ICs present in the unfractionated specimen. These results indicate that human AML stem cells are rare and enriched but not restricted to the phenotype associated with normal primitive hematopoietic cells. These results suggest a plasticity of the cancer stem cell phenotype that we believe has not been previously described.

A phase I study of the mammalian target of rapamycin inhibitor sirolimus and MEC chemotherapy in relapsed and refractory acute myelogenous leukemia.

PURPOSE: Inhibiting mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) blasts and leukemic stem cells may enhance their sensitivity to cytotoxic agents. We sought to determine the safety and describe the toxicity of this approach by adding the mTOR inhibitor, sirolimus (rapamycin), to intensive AML induction chemotherapy. EXPERIMENTAL DESIGN: We performed a phase I dose escalation study of sirolimus with the chemotherapy regimen MEC (mitoxantrone, etoposide, and cytarabine) in patients with relapsed, refractory, or untreated secondary AML. RESULTS: Twenty-nine subjects received sirolimus and MEC across five dose levels. Dose-limiting toxicities were irreversible marrow aplasia and multiorgan failure. The maximum tolerated dose (MTD) of sirolimus was determined to be a 12 mg loading dose on day 1 followed by 4 mg/d on days 2 to 7, concurrent with MEC chemotherapy. Complete or partial remissions occurred in 6 (22%) of the 27 subjects who completed chemotherapy, including 3 (25%) of the 12 subjects treated at the MTD. At the MTD, measured rapamycin trough levels were within the therapeutic range for solid organ transplantation. However, direct measurement of the mTOR target p70 S6 kinase phosphorylation in marrow blasts from these subjects only showed definite target inhibition in one of five evaluable samples. CONCLUSIONS: Sirolimus and MEC is an active and feasible regimen. However, as administered in this study, the synergy between MEC and sirolimus was not confirmed. Future studies are planned with different schedules to clarify the clinical and biochemical effects of sirolimus in AML and to determine whether target inhibition predicts chemotherapy response.

A phase I study of bexarotene, a retinoic X receptor agonist, in non-M3 acute myeloid leukemia.

PURPOSE: Bexarotene is a retinoic X receptor agonist that has been shown in vitro to inhibit growth and induce differentiation of myeloid leukemic cell lines. We therefore conducted a phase I dose escalation study to assess the maximum tolerated dose, toxicities, and activity of bexarotene in patients with non-M3 acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We enrolled patients with active non-M3 AML who had either relapsed or refractory disease or were not eligible for standard cytotoxic chemotherapy. Cohorts of three to six patients received escalating doses of daily oral bexarotene ranging from 100 to 400 mg/m(2) until evidence of disease progression or unacceptable adverse events occurred. RESULTS: Twenty-seven patients, with median age of 69 years (range, 51-82 years), were treated. Twenty-four (89%) patients had undergone prior chemotherapy. At the highest dose level tested (400 mg/m(2)), three of six patients had to reduce their dose of bexarotene due to grade 3 adverse events. The maximum tolerable dose of bexarotene was determined to be 300 mg/m(2). Clinical activity was manifested by 4 (15%) patients with reduction in bone marrow blasts to 1 year while taking bexarotene. Leukemic blast differentiation was suggested by the presence of the leukemic cytogenetic abnormality in mature circulating granulocytes and the occurrence of differentiation syndrome. CONCLUSIONS: The recommended dose of bexarotene for future studies is 300 mg/m(2)/d. Bexarotene is well tolerated in patients with non-M3 AML and has evidence of antileukemic activity.

Lestaurtinib (CEP701) is a JAK2 inhibitor that suppresses JAK2/STAT5 signaling and the proliferation of primary erythroid cells from patients with myeloproliferative disorders.

Recent studies have demonstrated that patients with myeloproliferative disorders (MPDs) frequently have acquired activating mutations in the JAK2 tyrosine kinase. A multikinase screen determined that lestaurtinib (formerly known as CEP-701) inhibits wild type JAK2 kinase activity with a concentration that inhibits response by 50% (IC(50)) of 1 nM in vitro. We hypothesized that lestaurtinib would inhibit mutant JAK2 kinase activity and suppress the growth of cells from patients with MPDs. We found that lestaurtinib inhibits the growth of HEL92.1.7 cells, which are dependent on mutant JAK2 activity for growth in vitro and in xenograft models. Erythroid cells expanded from primary CD34(+) cells from patients with MPDs were inhibited by lestaurtinib at concentrations of 100 nM or more in 15 of 18 subjects, with concomitant inhibition of phosphorylation of STAT5 and other downstream effectors of JAK2. By contrast, growth of erythroid cells derived from 3 healthy controls was not significantly inhibited. These results demonstrate that lestaurtinib, in clinically achievable concentrations, inhibits proliferation and JAK2/STAT5 signaling in cells from patients with MPDs, and therefore holds promise as a therapeutic agent for patients with these disorders.

Evidence of myeloid differentiation in non-M3 acute myeloid leukemia treated with the retinoid X receptor agonist bexarotene.

All-trans-retinoic acid has dramatically changed the treatment paradigm for acute promyelocytic leukemia, however, it has no significant activity in non-M3 acute myeloid leukemia (AML). In vitro, bexarotene, a retinoid X receptor agonist inhibits the proliferation of non-M3 AML cell lines and induces differentiation of leukemic blasts from patients. We hypothesized that there may be similar activity in patients with AML. We report on two patients with relapsed or refractory non-M3 AML treated with bexarotene monotherapy. After initiating treatment, both patients showed leukemic differentiation in their peripheral blood and reduction in bone marrow blasts to less than 5%. One patient had a significant improvement in her platelet count with loss of platelet transfusion needs. Differentiation syndrome occurred in one patient and was successfully treated with steroids and discontinuation of bexarotene. These data suggest that bexarotene has clinical activity in non-M3 AML and may be able to induce myeloid differentiation in vivo.

Neuromedin U: a Myb-regulated autocrine growth factor for human myeloid leukemias.

The c-myb proto-oncogene has been implicated in leukemogenesis, but possible mechanisms remain ill defined. To gain further insight to this process, we used transcript profiling in K562 cells expressing a dominant-negative Myb (MERT) protein. A total of 105 potential Myb gene targets were identified. Neuromedin U (NmU), a peptide affecting calcium transport, underwent the greatest expression change ( approximately 5-fold decrease). To verify a linkage between c-myb and NmU, their mRNA levels were quantitated using real-time polymerase chain reaction in primary acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), as well as normal hematopoietic cells. We found that c-myb was elevated in AML and ALL samples, but NmU expression was increased only in AML cells. Significantly, only AML cells expressed the cognate receptor of NmU, NMU1R, suggesting the presence of a novel autocrine loop. We examined this possibility in detail. Exogenous NmU "rescued" growth suppression in K562-MERT cells and stimulated the growth of primary AML cells. Short interfering RNA "knockdown" of NmU in K562 cells arrested cell growth. Exposing Indo-1-labeled K562 cells to NmU induced an intracellular Ca(++) flux consistent with engagement of the NMU1R. Combined, these results suggest that NmU expression is related to Myb and that the NmU/NMU1R axis constitutes a previously unknown growth-promoting autocrine loop in myeloid leukemia cells.