Multiple conformational states of the HPK1 kinase domain in complex with sunitinib reveal the structural changes accompanying HPK1 trans-regulation.

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.

Hematopoietic progenitor kinase 1 is a negative regulator of dendritic cell activation.

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 kinases that acts as a negative regulator of T cell functions through the AP-1, NFAT, and NFkappaB pathways. Using HPK1-deficient (HPK1(-/-)) mice, we report in this study a novel role for HPK1 in dendritic cells (DCs). Specifically, we observed that matured HPK1(-/-) bone marrow-derived DCs (BMDCs) are superior to their wild-type (WT) counterpart in stimulating T cell proliferation in vivo and in vitro. Several characteristics of HPK1(-/-) BMDCs may account for this enhanced activity: Matured HPK1(-/-) BMDCs express higher levels of costimulatory molecules CD80, CD86, and I-A(b) as well as produce more proinflammatory cytokines IL-12, IL-1beta, TNF-alpha, and IL-6 than their WT littermates. The role of HPK1 as a proapoptotic molecule was assessed post activation with LPS, and results indicated that HPK1(-/-) BMDCs are significantly resistant to LPS-induced apoptosis. Our results led us to investigate the role of HPK1(-/-) BMDCs in tumor immunotherapy. Using a s.c. murine model of Lewis Lung Carcinoma, we found that HPK1(-/-) BMDCs eliminate established s.c. Lewis Lung Carcinoma more efficiently than their WT counterpart. Our data reveal a novel role for HPK1 as a negative regulator of DC functions, identifying its potential as a molecular target for DC-based immunotherapy against cancers.

Hematopoietic progenitor kinase 1 is a critical component of prostaglandin E2-mediated suppression of the anti-tumor immune response.

Lung cancer is the leading cause of cancer-related mortality in the world, resulting in over a million deaths each year. Non-small cell lung cancers (NSCLCs) are characterized by a poor immunogenic response, which may be the result of immunosuppressive factors such as prostaglandin E2 (PGE(2)) present in the tumor environment. The effect of PGE(2) in the suppression of anti-tumor immunity and its promotion of tumor survival has been established for over three decades, but with limited mechanistic understanding. We have previously reported that PGE(2) activates hematopoietic progenitor kinase 1 (HPK1), a hematopoietic-specific kinase known to negatively regulate T-cell receptor signaling. Here, we report that mice genetically lacking HPK1 resist the growth of PGE(2)-producing Lewis lung carcinoma (LLC). The presence of tumor-infiltrating lymphocytes (TILs) and T-cell transfer into T cell-deficient mice revealed that tumor rejection is T cell mediated. Further analysis demonstrated that this may be significantly due to the ability of HPK1 (-/-) T cells to withstand PGE(2)-mediated suppression of T-cell proliferation, IL-2 production, and apoptosis. We conclude that PGE(2) utilizes HPK1 to suppress T cell-mediated anti-tumor responses.

A perspective on HPK1 as a novel immuno-oncology drug target.

In this perspective review, the role Hematopoietic Progenitor Kinase 1 (HPK1) in tumor immunity will be reviewed, with special emphasis on how T cells are negatively-regulated at different junctures of cancer-immunity cycle by this regulatory kinase. The review will highlight the strengths and weaknesses of HPK1 as a candidate target for novel immuno-oncology (IO) drug development that is centered on the use of small molecule kinase inhibitor to modulate the immune response against cancer. Such a therapeutic approach, if proven successful, could supplement the cancer cell-centric standard of care therapies in order to fully meet the therapeutic needs of cancer patients.

HPK1 Influences Regulatory T Cell Functions.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of TCR-initiated signal transduction. Both the HPK1-/- mice and the genetically engineered mice with a point mutation that disrupts the catalytic activity of HPK1 possess enhanced antitumor immunity, especially when these mice are treated with anti-PD-L1 immune checkpoint Ab. Because CD4+FOXP3+ regulatory T cells (Tregs) play an important role in suppressing tumor immunity, we investigated whether the loss of HPK1 expression could result in the reduction of Treg functions. We found that the number of HPK1-/- Tregs is elevated relative to the number found in wild-type C57/BL6 mice. However, HPK1-/- Tregs lack the ability to carry out effective inhibition of TCR-induced proliferative responses by effector T cells. Furthermore, HPK1-/- Tregs respond to TCR engagement with an elevated and sustained Erk MAPK and p65/RelA NF-κB phosphorylation in comparison with wild-type Tregs. Also, a multiplex cytokine analysis of HPK1-/- Tregs revealed that they demonstrate an aberrant cytokine expression profile when stimulated by anti-CD3ε and anti-CD28 crosslinking, including the uncharacteristic expression of IL-2 and antitumor proinflammatory cytokines and chemokines such as IFN-γ, CCL3, and CCL4. The aberrant HPK1-/- phenotype observed in these studies suggests that HPK1 may play an important role in maintaining Treg functions with wider implications for HPK1 as a novel immunotherapeutic target.

The Structure of HPK1 Kinase Domain: To Boldly Go Where No Immuno-Oncology Drugs Have Gone Before.

In this issue of Structure, Wu et al. (2018) report several apo and small-molecule inhibitor-bound structures of the kinase domain of hematopoietic progenitor kinase 1, a ser/thr kinase that functions as an inhibitor of T cell activation. The studies reveal that the HPK1 kinase domain exists as a domain-swapped dimer.

HPK1 as a novel target for cancer immunotherapy.

Identifying the appropriate drug targets for the development of a novel anti-tumor immunotherapy is one of the most risky steps in the drug development cycle. We have identified a hematopoietic cell-restricted serine/threonine kinase, hematopoietic progenitor kinase 1 (HPK1), as a possible target for therapeutic intervention. Targeted disruption of HPK1 alleles confers T cells with an elevated Th1 cytokine production in response to TCR engagement. HPK1 (-/-) T cells proliferate more rapidly than the haplotype-matched wild-type counterpart and are resistant to prostaglandin E2 (PGE(2))-mediated suppression. Most strikingly, mice that received adoptive transfer of HPK1 (-/-) T cells became resistant to lung tumor growth. Also, the loss of HPK1 from dendritic cells (DCs) endows them with superior antigen presentation ability, enabling HPK1 (-/-) DCs to elicit a more potent anti-tumor immune response when used as cancer vaccine. It is probable that blocking the HPK1 kinase activity with a small molecule inhibitor may activate the superior anti-tumor activity of both cell types, resulting in a synergistic amplification of anti-tumor potential. Given that HPK1 is not expressed in any major organs, it is less likely that an inhibitor of HPK1 kinase activity would cause any serious side effects.

Detecting tyrosine-phosphorylated proteins by Western blot analysis.

The development of monoclonal antibodies (mAbs) that recognize nearly all of the phosphorylated tyrosine residues, irrespective of the surrounding sequences, enables researchers to detect the phosphorylation state of proteins through the use of anti-phosphotyrosine western blotting. The availability of this simple, reliable, nonradioactive and yet sensitive method created a boom in signal transduction research. While the methodology of how to perform an anti-phosphotyrosine western blot remains unchanged since the procedure became widely used in the early part of 1990s, steady improvements in reagents and detection technologies have allowed researchers to detect tyrosine phosphorylation quantitatively, at unprecedented sensitivity. In addition to the improvements in the western blot-based systems, powerful new phosphotyrosine detection platforms, based on proteomic technologies, are emerging rapidly. This unit will describe in detail the steps needed to perform the standard anti-phosphotyrosine western blot analysis.

Prostaglandin E2 activates HPK1 kinase activity via a PKA-dependent pathway.

Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that the immunosuppressive eicosanoid, prostaglandin E(2) (PGE(2)), is capable of activating HPK1 in T cells. In this report, we demonstrate that unlike the TCR-induced activation of HPK1 kinase activity, the induction of HPK1 catalytic activity by PGE(2) does not require the presence of phosphotyrosine-based signaling molecules such as Lck, ZAP-70, SLP-76, and Lat. Nor does the PGE(2)-induced HPK1 activation require the intermolecular interaction between its proline-rich regions and the SH3 domain-containing adaptor proteins, as required by the signaling from the TCR to HPK1. Instead, our study reveals that PGE(2) signal to HPK1 via a 3' -5 '-cyclic adenosine monophosphate-regulated, PKA-dependent pathway. Consistent with this observation, changing the serine 171 residue that forms the optimal PKA phosphorylation site within the "activation loop" of HPK1 to alanine completely prevents this mutant from responding to PGE(2)-generated stimulation signals. Moreover, the inability of HPK1 to respond to PGE(2) stimulation in PKA-deficient S49 cells further supports the importance of PKA in this signaling pathway. We speculate that this unique signaling pathway enables PGE(2) signals to engage a proven negative regulator of TCR signal transduction pathway and uses it to inhibit T cell activation.

Hematopoietic progenitor kinase 1 (HPK1) negatively regulates prostaglandin E2-induced fos gene transcription.

Prostaglandin E(2) (PGE(2)) is the predominant eicosanoid product released by macrophages at the site of inflammation. Binding of PGE(2) to its cognate 7 transmembrane-spanning G protein-coupled receptors (GPCRs) activates signaling pathways, leading to the synthesis of the Fos transcription factor. Because the Ste20 serine/threonine protein kinase (S/TPK) is a critical signal transducer for the G protein-coupled pheromone receptor in Saccharomyces cerevisiae, we postulated that the PGE(2) GPCRs may activate one of the Ste20 mammalian orthologs. We demonstrate here that the catalytic activity of a hematopoietic cell-restricted, Ste20-related S/TPK, HPK1, is positively regulated by exposure to physiological concentrations of PGE(2). Furthermore, ectopic expression studies implicated HPK1 as a negative regulator of PGE(2)-induced transcription of the fos gene. Our data suggest that PGE(2)-induced activation of HPK1 may represent a novel negative regulatory pathway capable of modulating PGE(2)-mediated gene transcription.

A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling.

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.